Anti-cd20 antibody and uses thereof

ABSTRACT

Provided herein are anti-CD20 antibodies and uses thereof for treatment and diagnosis. Also provided are CD20 antigens for the production of anti-CD20 antibodies and methods of generating anti-CD20 antibodies using the CD20 antigens.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.16/970,805, filed Aug. 18, 2020, which is the National Stage Applicationof PCT/US2019/018535, filed Feb. 19, 2019, which claims priority to U.S.Provisional Application No. 62/633,034, filed February 2018, the entirecontents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 22, 2019, isnamed 115872-0538 SL.txt and is 36,516 bytes in size.

BACKGROUND OF THE INVENTION

The majority of adult B-cell malignancies, including acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin'slymphoma, are incurable despite currently available therapies. However,adoptive therapy with genetically engineered chimeric antigen receptor(CAR) T cells is one approach that has been recently highly successfulin patients with acute lymphocytic leukemia (ALL) and has also generatedresponses in patients with chronic lymphocytic leukemia (CLL) and B cellnon-Hodgkin lymphoma (NHL), although less profound (Park et al. (2016)Blood 127(26):3312-20; Davila et al. (2014) Science TranslationalMedicine 6(224):224ra25; Davila et al. (2016) Int J Hematol.104(1):6-17; Turtle et al. (2016) Science Translational Medicine8(355):355ra116).

Adoptive cell transfer (ACT) is a form of immunotherapy that involvesthe transfer of immune cells with antitumor activity into patients. ACTtypically involves isolation of lymphocytes with antitumor activity froma patient, culturing the lymphocytes in vitro to expand the population,and then infusing the lymphocytes into the cancer-bearing host.Lymphocytes used for adoptive transfer can either be derived from thestroma of resected tumors (e.g., tumor infiltrating lymphocytes), fromthe lymphatics or lymph nodes, or from the blood. In some cases, theisolated lymphocytes are genetically engineered to express anti-tumor Tcell receptors (TCRs) or chimeric antigen receptors (CARs). Thelymphocytes used for infusion can be isolated from a donor (allogeneicACT), or from the cancer-bearing host (autologous ACT). Immunotherapy isa targeted therapy that has the potential to provide for the treatmentof cancer.

However, malignant cells adapt to generate an immunosuppressivemicroenvironment to protect themselves from immune recognition andelimination. This “hostile” tumor microenvironment poses a challenge tomethods of treatment involving stimulation of an immune response, suchas targeted T cell therapies. Accordingly, novel therapeutic strategiesfor treating neoplasia are needed.

SUMMARY OF THE INVENTION

Provided herein, in certain embodiments, are anti-CD20 antibodies andCD20 antigen binding fragments thereof. In some embodiments, theanti-CD20 antibodies and CD20 antigen binding fragments thereof arespecific for canine CD20. In some embodiments, the isolated antibody oran antigen binding portion thereof that specifically binds to a canineCD20 cyclic peptide having the sequence of SEQ ID NO: 20, wherein thecysteine at position 7 of SEQ ID NO: 20 forms a disulfide bond with thecysteine at position 23 of SEQ ID NO: In some embodiments, the antibodyor an antigen binding portion thereof binds to the canine CD20 cyclicpeptide at a higher affinity than a linear peptide having the sequenceof SEQ ID NO: 21. In some embodiments, the antibody or an antigenbinding portion thereof comprises a heavy chain variable domain (VH)comprising SEQ ID NO: 4, or is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the antibodyor an antigen binding portion thereof comprises a VH complementaritydetermining region (CDR)1 of SEQ ID NO: 40, or is at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:40. In some embodiments, the antibody or an antigen binding portionthereof comprises a VH CDR2 of SEQ ID NO: 42, or is at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:42. In some embodiments, the antibody or an antigen binding portionthereof comprises a VH CDR3 consisting of a threonine (T) residue. Insome embodiments, the antibody or an antigen binding portion thereofcomprises a light chain variable domain (LH) comprising SEQ ID NO: 6, oris at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQID NO: 6. In some embodiments, the antibody or an antigen bindingportion thereof comprises a VL CDR1 of SEQ ID NO: 46, or is at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 46. In some embodiments, the antibody or an antigen bindingportion thereof comprises a VL CDR2 of SEQ ID NO: 48 or a VL CDR2 havingat least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 48. In some embodiments, the antibodyor an antigen binding portion thereof comprises a VL CDR3 of SEQ ID NO:50 or a VL CDR3 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 50. In someembodiments, the isolated antibody or an antigen binding portion thereofis a full length antibody, a Fab fragment, a F(ab′)2 fragment, or asingle chain variable fragment (scFV). In some embodiments, the isolatedantibody or an antigen binding portion thereof is a scFv. In someembodiments, the isolated antibody or an antigen binding portion thereofcomprises SEQ ID NO: 8, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 8. In some embodiments, the isolatedantibody or an antigen binding portion thereof is a chimeric antigenreceptor (CAR).

Provided herein, in certain embodiments are fusion proteins comprisingthe isolated antibody or an antigen binding portion thereof providedherein where the fusion protein comprises a scFv-Fc fusion protein,immunoconjugate, or bispecific antibody. In some embodiments, the fusionprotein comprises a second component selected from the group consistingof a cytotoxin, a detectable label, a radioisotope, a therapeutic agent,a liposome, a nanoparticle, a binding protein, or an antibody.

Provided herein, in certain embodiments, are nucleic acids encoding theisolated an antibody or antigen binding portion thereof provided herein.Also provided herein, in certain embodiments, are expression vectorscomprising a nucleic acid provided herein. Also provided herein, incertain embodiments, are cells comprising a nucleic acid or vectorprovided herein. In some embodiments, the cell is an immune cell. Insome embodiments, the immune cell is a T-cell or natural killer (NK)cell.

Provided herein, in certain embodiments, are methods for the productionof anti-CD20 antibodies and CD20 antigen binding fragments thereof. Insome embodiments, the anti-CD20 antibody or antigen binding fragmentthereof is labeled with a detectable moiety. In some embodiments, theanti-CD20 antibody or antigen binding fragment thereof is a fullimmunoglobulin, a heavy chain variable region (VH), a light chainvariable region (VL) or a single-chain variable fragment (scFv).

Provided herein, in certain embodiments, are chimeric T cell receptorscomprising one or more complementarity determining regions (CDR) of ananti-CD20 antibody or antigen binding fragment thereof provided herein.Provided herein, in certain embodiments, are chimeric T cell receptorscomprising CDR1, CDR2, and/or CDR3 of an anti-CD20 antibody or antigenbinding fragment thereof provided herein. Provided herein, in certainembodiments, are chimeric T cell receptors comprising the light chain oran antigen-binding portion thereof of an anti-CD20 antibody providedherein. Provided herein, in certain embodiments, are chimeric T cellreceptors comprising the heavy chain or an antigen-binding portionthereof of an anti-CD20 antibody provided herein. Provided herein, incertain embodiments, are chimeric T cell receptors comprising a heavychain or an antigen-binding portion thereof and a light chain or anantigen-binding portion thereof of an anti-CD20 antibody providedherein. In some embodiments, the chimeric T cell receptor comprises aco-stimulatory portion. In some embodiments, the co-stimulatory portionis a human, rodent, or canine co-stimulatory protein. In someembodiments, the chimeric T cell receptor comprises a CD3 zeta chain. Insome embodiments, the co-stimulatory portion is a human, rodent, orcanine CD3 zeta chain.

In some embodiments, a chimeric antigen receptor provided hereincomprises (i) antigen binding portion comprising the isolated antibodyor antigen binding portion thereof provided here or a fusion proteinprovided herein; (ii) a transmembrane portion; and (iii) a cytoplasmicsignaling portion. In some embodiments, the antigen binding portioncomprises a heavy chain variable domain (VH) comprising SEQ ID NO: 4, oris at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQID NO: 4 and/or a light chain variable domain (LH) comprising SEQ ID NO:6, or is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 6. In some embodiments, the antigen binding portion comprises(a) a heavy chain variable domain (VH) comprising (i) a variable heavychain (VH) complementarity determining region (CDR) 1 of SEQ ID NO: 40,or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 40; (ii) a VH CDR2 of SEQ ID NO: 42, or at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 42; and/or (iii) a VH CDR3 consisting of a threonine (T)residue; and/or (b) a light chain variable domain (LH) comprising (i) alight chain variable domain (VL) complementarity determining region(CDR) 1 of SEQ ID NO: 46, or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 46; (b) a VL CDR2 of SEQID NO: 48, or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical to SEQ ID NO: 48; and/or (c) a VL CDR3 of SEQ ID NO:50, or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 50. In some embodiments, the antigen bindingportion comprises SEQ ID NO: 8, or is at least 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the chimericantigen receptor comprises a CD8 leader sequence, a CD28 costimulatorydomain, a CD3-chain, a 4-1BBL costimulatory domain, a PD1-DNR domain, aCD34 domain, or any combination thereof.

Provided herein, in certain embodiments, are cells expressing thechimeric antigen receptor provided herein. In some embodiments, the cellis a T cell or natural killer (NK) cell.

Provided herein, in certain embodiments, are methods for the productionof chimeric T cell receptors comprising an anti-CD20 antibody or antigenbinding fragment thereof provided herein.

Provided herein, in certain embodiments, are pharmaceutical compositionscomprising an antigen-binding protein or fragment or derivative thereofprovided herein or a fusion protein provided herein; and aphysiologically acceptable diluent, excipient or carrier.

Provided herein, in certain embodiments, are methods of treatment usingthe anti-CD20 antibodies or antigen binding fragments thereof providedherein.

Provided herein, in certain embodiments, are methods for inhibitingtumor growth or metastasis comprising contacting a tumor cell with aneffective amount of the antibody or an antigen binding portion thereofprovided herein. In some embodiments, the tumor cell is a canine tumorcell.

Provided herein, in certain embodiments, are methods for treating acondition mediated by B-cells in a subject in need thereof comprisingadministering an effective amount of the antibody or an antigen bindingportion thereof provided herein. In some embodiments, the conditionmediated by B-cells is a B cell lymphoma. In some embodiments, thecondition mediated by B-cells is an immune mediated disease. In someembodiments, the immune mediated disease is an autoimmune disease. Insome embodiments, the autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, systemic lupus erythematosus (SLE),Sjogren's syndrome, vasculitis, multiple sclerosis, Graves' disease,idiopathic thrombocytopenia, dermatomyositis, immune mediatedthrombocytopenia, polymyocytosis, pemphigus, immune mediated hemolyticanemia and bullous pemphigoid.

Provided herein, in certain embodiments, are methods for treatmentcomprising isolating T-cells from a subject, transfecting the T-cellswith a vector comprising a nucleic acid encoding an antibody or anantigen binding portion thereof provided herein, and administering thetransfected T-cells to the subject. In some embodiments, the subject isa canine subject.

Provided herein, in certain embodiments, are uses of an antibody or anantigen binding fragment thereof that specifically binds to a canineCD20 cyclic peptide having the sequence of SEQ ID NO: 20, wherein thecysteine at position 7 of SEQ ID NO: 20 forms a disulfide bond with thecysteine at position 23 of SEQ ID NO: 20 in the preparation of amedicament for the treatment or prevention of a condition mediated byB-cells in a subject in need thereof, wherein the cysteine at position 7of SEQ ID NO: 20 forms a disulfide bond with the cysteine at position 23of SEQ ID NO: 20. In some embodiments, the condition mediated by B-cellsis a B cell lymphoma or leukemia. In some embodiments, the conditionmediated by B-cells is an immune mediated disease. In some embodiments,the immune mediated disease is an autoimmune disease. In someembodiments, the autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, systemic lupus erythematosus (SLE),Sjogren's syndrome, vasculitis, multiple sclerosis, Graves' disease,idiopathic thrombocytopenia, dermatomyositis, immune mediatedthrombocytopenia, polymyocytosis, pemphigus, immune mediated hemolyticanemia and bullous pemphigoid. In some embodiments, the isolatedantibody or an antigen binding portion thereof comprises a heavy chainvariable domain (VH) comprising SEQ ID NO: 4, or is at least 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In someembodiments, the isolated antibody or an antigen binding portion thereofcomprises a light chain variable domain (LH) comprising SEQ ID NO: 6, oris at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6. In some embodiments, the isolated antibody or an antigen bindingportion thereof is a full length antibody, a Fab fragment, a F(ab′)2fragment, or a single chain variable fragment (scFV). In someembodiments, the antibody is a scFv. In some embodiments, the isolatedantibody or an antigen binding portion thereof comprises SEQ ID NO: 8,or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 8.

Provided herein, in certain embodiments, are antigens for the productionanti-CD20 antibodies or antigen binding fragments thereof providedherein. In some embodiments, provided is a canine CD20 cyclic peptidecomprising a canine CD20 epitope having the sequence of SEQ ID NO: 20,wherein the cysteine at position 7 of SEQ ID NO: 20 forms a disulfidebond with the cysteine at position 23 of SEQ ID NO: 20. In someembodiments, the canine CD20 cyclic peptide further comprises a carrierprotein. In some embodiments, the carrier protein is KLH. In someembodiments, the carrier protein is conjugated to a linker. In someembodiments, the linker is a sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate linking group. In someembodiments, the canine CD20 cyclic peptide comprises a peptide spacerbetween the canine CD20 epitope and the carrier protein. In someembodiments, the peptide spacer comprises the sequence of SEQ ID NO:4.Also provided herein are methods of producing antibodies using a canineCD20 cyclic peptide provided herein. Also provided herein are methods ofstimulating of an immune response in vitro or in vivo using a canineCD20 cyclic peptide provided herein.

Provided herein, in certain embodiments, are methods for the detectionof CD20 protein or an extracellular domain of a CD20 protein in abiological sample with any of the anti-CD20 antibodies or antigenbinding fragments thereof provided herein. In some embodiments, themethods for detecting a CD20 protein or an extracellular domain of aCD20 protein in a biological sample, comprises contacting a biologicalsample with the antibody or an antigen binding portion thereof providedherein. In some embodiments, the biological sample is a biopsy, tissue,blood, serum, plasma, or lymphatic fluid sample. In some embodiments,the biological sample comprises cells that express a CD20 protein or afragment thereof or is a cell lysate prepared from cells that express aCD20 protein or a fragment thereof. In some embodiments, the biologicalsample comprises B cells or a B cell lysate.

Also provided are compositions, kits, and methods of treatment relatingto antigen-binding proteins that bind to a canine CD20 antigenic peptide(K9CD20). The antigen-binding proteins disclosed herein demonstrateimproved specificity for an epitope comprised in the extracellulardomain of canine CD20 protein, and can mediate killing of B-celllymphoma in vitro and in vivo. In some embodiments, a kits for detectinga CD20 protein or an extracellular domain of a CD20 protein in abiological sample comprises an antibody or an antigen binding portionthereof provided herein and at least one reagent for the detection ofthe antibody or antigen binding portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure human CD20 (SEQ ID NO: 59)_(adaptedfrom Ernst J. A. et al. Biochemistry 44: 15150 (2005)).

FIG. 2 illustrates the extracellular portion of human CD20 (SEQ ID NO:60).

FIG. 3 . illustrates CD20 extracellular sequences in canine (SEQ ID NO:1), human (SEQ ID NO: 2), and mouse (SEQ ID NO: 3) for epitope design.The highlighted portion in the human sequence is the rituximab epitope.The highlighted portion of the canine CD20 represents the epitope thatwas used in the present disclosure to generate canine CD20 antibodies.The two cysteines in the canine epitope are cyclized through cysteineside chains.

FIG. 4 illustrates canine CD20 epitope design comprising the canine CD20epitope (SEQ ID NO: 20) and the space sequence (SEQ ID NO: 52). FIG. 4discloses the full-length sequence as SEQ ID NO: 61.

FIG. 5 illustrates an exemplary schema for conjugation of the preparedcanine CD20 antigen comprising the canine CD20 epitope (SEQ ID NO: 20)and the space sequence (SEQ ID NO: 52) to carrier protein KLH. FIG. 5discloses the full-length sequence as SEQ ID NO: 61.

FIG. 6A and FIG. 6B each illustrate one replica of ELISA screening ofall bleeds collected from four rats immunized with a synthetic canineCD20 antigenic peptide, according to one embodiment of the presentdisclosure.

FIG. 7 illustrates a plot of ELISA OD values versus sampleconcentrations for serial dilutions of serum collected from a ratimmunized with a synthetic canine CD20 antigenic peptide, according toone embodiment of the present disclosure.

FIG. 8 illustrates anti-canine CD20 specificity of antibodies producedby animals immunized with a synthetic canine CD20 antigenic peptide,according to one embodiment of the present disclosure.

FIG. 9 illustrates anti-canine CD20 specificity of antibodies producedby animals immunized with a synthetic canine CD20 antigenic peptide forcells expressing canine CD20 (K9 3T3) versus normal dog cells.

FIG. 10 illustrates that human T cells targeted to K9CD20 are functionaland cytotoxic. FIG. 10A shows the transduction efficiency inK9CD20-targeted chimeric antigen receptor (CAR) T cells assessed bycoexpression of L-NGFR. FIG. 10B shows the results of a Cr51 cytotoxicrelease assay in EL4 tumor cells. FIG. 10C shows the results of a Cr51cytotoxic release assay in NALM6 or NALM6-K9CD20 tumor cells.

FIG. 11 illustrates human T cells expressing K9CD20-targeted CAR arefunctional in vivo.

FIG. 12 shows the results of a Cr51 cytotoxic release assay in NALM6 orNALM6-K9CD20 tumor cells.

FIG. 13 shows the results of a Cr51 cytotoxic release assay in NALM6 orNALM6-K9CD20 tumor cells.

FIG. 14 illustrates schematics of CAR cloning constructs. FIG. 14Aillustrates the cloning construct for the light and heavy chain variableregions of the rat anti-canine CD20.

FIG. 14B illustrates the anti-K9CD20 scFv construct. FIG. 14B discloses“(G3S)4” as SEQ ID NO: 62. FIG. 14C illustrates the SFG-anti-K9CD20 CARLNGFR construct. FIG. 14C discloses “(G35)4” as SEQ ID NO: 62. FIG. 14Dillustrates the SFG-K9CD20-dsRed construct. FIG. 14E illustrates theSFG-K27-CAR construct. FIG. 14E discloses “(G3S)4” as SEQ ID NO: 62.FIG. 14F illustrates the SFG-K36-CAR construct. FIG. 14F discloses“(G35)4” as SEQ ID NO: 62. FIG. 14G illustrates the SFG-K9CD34t-K27construct. FIG. 14G discloses “(G35)4” as SEQ ID NO: 62. FIG. 14Hillustrates the SFG-K9CD34t-K36 construct. FIG. 14H discloses “(G35)4”as SEQ ID NO: 62. FIG. 14I illustrates the K27/PD1-DNR construct. FIG.14I discloses “(G35)4” as SEQ ID NO: 62. FIG. 14J illustrates SFG vectorbackbone. FIG. 14K illustrates the SFG-PmlI-αk9CD2028z-LNGFR-BamHIconstruct.

FIG. 15 illustrates a canine treatment timeline

FIG. 16 illustrates a schematic of the use of K9CD20-targeted CAR canineT cells for the treatment of B cell malignancies. CAR function can bepotentiated by co-expression of PD1-DNR or 41BBL. 41BBL can interact incis upon activation and upregulation of 41BB on CART cells (auto:autocostimulation) and in trans by interacting with 41BB on other CAR orendogenous T cells (trans: transcostimulation). PD1-DNR interacts withPDL-1 expressed on tumor cells and offsets the inhibition of CAR T cellfunction.

FIG. 17 illustrates the flow cytometry data for 3T3-K9CD20 cells stainedwith the supernatant from hybridoma clone 5B3. FIG. 17A illustrates theforward scatter and side scatter plot. FIG. 17B illustrates thehistogram for FITC-A.

FIG. 18 illustrates the flow cytometry data for 3T3-K9CD20 cells stainedwith the supernatant from hybridoma clone 10C10. FIG. 18A illustratesthe forward scatter and side scatter plot. FIG. 18B illustrates thehistogram for FITC-A.

FIG. 19 illustrates the flow cytometry data for 3T3-K9CD20 cells stainedwith the supernatant from hybridoma clone 18F6. FIG. 19A illustrates theforward scatter and side scatter plot. FIG. 19B illustrates thehistogram for FITC-A.

FIG. 20 illustrates the flow cytometry data for 3T3-K9CD20 cells stainedwith the supernatant from hybridoma clone 7A7 K9 (negative control).FIG. 20A illustrates the forward scatter and side scatter plot. FIG. 20Billustrates the histogram for FITC-A.

FIG. 21 illustrates an exemplary schema for solid phase peptidesynthesis and protecting group strategy. FIG. 21 discloses SEQ ID NO:61.

FIG. 22 illustrates an exemplary schema for solid phase peptidesynthesis and the selective deprotection of cysteine side chains. FIG.22 discloses SEQ ID NO: 61, 61, and 61.

FIG. 23 illustrates an exemplary schema for solid phase peptidesynthesis and conjugation to KLH. FIG. 23 discloses SEQ ID NO: 61, 61,and 61.

FIG. 24 illustrates flow cytometry data for Canine PBMCs cells stainedwith CD21 and anti-CD20 hybridoma clone 18F6. The top panels show cellsstained with CD21 (PE/FL2-H) and no stain on FL1-H. The bottom panelsshow cells stained with CD21 (PE/FL2-H) and the antibody from hybridomaclone 18F6 (18F6+Goat anti rat IgG-FITC/FL1-H). The left panels of eachset illustrate the forward scatter and side scatter plots (FSC v. SSC),and the right panels of each set illustrate the fluorescence plots.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present disclosure. All thevarious embodiments of the present disclosure will not be describedherein. Many modifications and variations of the present disclosure canbe made without departing from its spirit and scope, as will be apparentto those skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present disclosure, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled.

It is to be understood that the present disclosure is not limited toparticular uses, methods, reagents, compounds, compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In addition, where features or aspects of the present disclosure aredescribed in terms of Markush groups, those skilled in the art willrecognize that the present disclosure is also thereby described in termsof any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

I. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the presentdisclosure. In the description that follows, certain conventions will befollowed regarding the usage of terminology. Generally, terms usedherein are intended to be interpreted consistently with the meaning ofthose terms as described below and as they are known to those of skillin the art.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

As used herein, the term “about” means that a value can vary +/−20%,+/−15%, +/−10% or +/−5% and remain within the scope of the presentdisclosure. For example, “a concentration of about 200 IU/mL”encompasses a concentration between 160 IU/mL and 240 IU/mL.

As used herein, the term “administration” of an agent to a subjectincludes any route of introducing or delivering the agent to a subjectto perform its intended function. Administration can be carried outlocally or systemically (e.g., enteral or parenteral administration).Administration can be carried out by any suitable route, includingintravenously, intramuscularly, intraperitoneally, orally, topically,intradermally, transdermally, intratumorally, intraocularly,intracerebrally, epidurally, intrathecally intranasally,intratracheally, intraosseously, epicutaneously, or subcutaneously.Administration includes self-administration and the administration byanother.

As used herein, the term “amino acid” refers to naturally occurring andnon-naturally occurring amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally encoded amino acids are the 20 commonamino acids (alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine.Amino acid analogs refer to agents that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,such as, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. In some embodiments, amino acidsforming a polypeptide are in the D form. In some embodiments, the aminoacids forming a polypeptide are in the L form. In some embodiments, afirst plurality of amino acids forming a polypeptide are in the D formand a second plurality are in the L form.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter code.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to naturally occurring amino acid polymers aswell as amino acid polymers in which one or more amino acid residues isa non-naturally occurring amino acid, e.g., an amino acid analog. Theterms encompass amino acid chains of any length, including full lengthproteins, wherein the amino acid residues are linked by covalent peptidebonds.

As used herein, a “control” is an alternative sample used in anexperiment for comparison purpose. A control can be “positive” or“negative.” For example, where the purpose of the experiment is todetermine a correlation of the efficacy of a therapeutic agent for thetreatment for a particular type of disease, a positive control (acomposition known to exhibit the desired therapeutic effect) and anegative control (a subject or a sample that does not receive thetherapy or receives a placebo) are typically employed.

As used herein, an “antigen-binding protein” is a protein or polypeptidethat comprises an antigen-binding region or antigen-binding portion thathas a strong affinity for another molecule to which it binds (antigen).Antigen-binding proteins encompass antibodies, antibody fragments,antibody derivatives, antibody analogs, fusion proteins, and antigenreceptors including chimeric antigen receptors (CARs). Anantigen-binding protein or fragment or derivative thereof optionallycomprises a scaffold or framework portion that allows theantigen-binding portion to adopt a conformation that promotes binding ofthe antigen-binding protein to the antigen. The antigen-binding proteincan comprise, for example, an alternative protein scaffold or artificialscaffold with grafted CDRs or CDR derivatives. Such scaffolds include,but are not limited to, antibody-derived scaffolds comprising mutationsintroduced to, for example, stabilize the three-dimensional structure ofthe antigen-binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Komdorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129 and Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronectin components as a scaffold.

In some embodiments, an antigen-binding protein or fragment orderivative thereof or fusion protein thereof has a single binding site.In some embodiments, an antigen-binding protein or fragment orderivative thereof or fusion protein thereof has more than one bindingsite. In some embodiments where there is more than one binding site, thebinding sites are identical to one another. In some embodiments wherethere is more than one binding site, the binding sites are different.For example, a naturally occurring human immunoglobulin typically hastwo identical binding sites, while a “bispecific antibody” or“bifunctional antibody” has two different binding sites.

As used herein, an “antibody” and “antibodies” refer to antigen-bindingproteins that arise in the context of the immune system. The term“antibody” as referred to herein includes whole, full length antibodiesand any fragment or derivative thereof in which the “antigen-bindingportion” or “antigen-binding region” or single chains thereof areretained. A naturally occurring “antibody” (immunoglobulin) is aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain is comprisedof a heavy chain variable region (abbreviated herein as VH) and a heavychain constant region. Heavy chains are classified as mu, delta, gamma,alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG,IgA, and IgE, respectively. The heavy chain constant region is comprisedof three domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRsarranged from amino terminus to carboxyl-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chainsform two regions: the Fab (fragment, antigen binding) region, alsoreferred to as the variable (Fv) region, and the Fc (fragment,crystallizable) region. The variable regions (Fv) of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant (Fc) regions of the antibodies can mediate the binding to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. The term “Fc” as used herein includes native andmutant forms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns. One suitable Fc polypeptide is derived from the human IgG1antibody.

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity orrecombinantly produced. If such antibodies are subjected to affinitymaturation, they can be enriched for a particular antigenic specificity.Such affinity matured preparations of antibodies usually are made ofless than about 10% of antibodies having specific binding activity forthe particular antigen. Subjecting these preparations to several roundsof affinity maturation can increase the proportion of antibody havingspecific binding activity for the antigen. Antibodies prepared in thismanner are often referred to as “affinity matured.”

Fragments, derivatives, or analogs of antigen-binding proteins such asantibodies can be readily prepared using techniques well-known in theart. The term “fragment” as used herein refers to a polypeptide that hasan amino-terminal and/or carboxyl-terminal deletion as compared to acorresponding full-length antigen-binding protein. Examples of fragmentsof antigen-binding proteins encompassed within the term “fragments”include a Fab fragment; a monovalent fragment consisting of the VL, VH,CL and CH1 domains; a F(ab′)2 fragment; a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the VH and CH1 domains; a Fv fragment consistingof the VL and VH domains of a single arm of an antibody; a dAb fragment(Ward et al., 1989, Nature, 341:544-546), which consists of a VH domain;an isolated complementarity determining region (CDR); and a single chainvariable fragment (scFv). A “derivative” of an antigen-binding proteinis a polypeptide (e.g., an antibody) that has been chemically modified,e.g., via conjugation to another chemical moiety (such as, for example,polyethylene glycol or albumin, e.g., human serum albumin),phosphorylation, and/or glycosylation.

As used herein, a “scFv” is a monovalent molecule that can be engineeredby joining, using recombinant methods, the two domains of the Fvfragment, VL and VH, by a synthetic linker that enables them to be madeas a single protein chain (see e.g., Bird et al., 1988, Science,242:423-426; and Huston et al., 1988, Proc. Natl. Acad. Sci.85:5879-5883). Such single chain antigen-binding peptides are alsointended to be encompassed within the term “antigen-binding portion.”These antibody fragments are obtained using conventional techniquesknown to those of skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. An exemplary scFvmolecule is provided below as SEQ ID NO: 10, which contains a VL segmentrepresented as SEQ ID NO: 6 and a VH segment represented as SEQ ID NO: 4connected by a linker sequence represented as SEQ ID NO: 10.

As used herein, “monoclonal antibody” refers to a population ofidentical antibodies, meaning that each individual antibody molecule ina population of monoclonal antibodies is identical to the others. Thisproperty is in contrast to that of a polyclonal population ofantibodies, which contains antibodies having a plurality of differentsequences. Monoclonal antibodies can be produced by a number ofwell-known methods (Smith et al. (2004) J. Clin. Pathol. 57: 912-917;and Nelson et al. (2000) J Clin Pathol, 53: 111-117). For example,monoclonal antibodies can be produced by immortalization of a B cell,for example through fusion with a myeloma cell to generate a hybridomacell line or by infection of B cells with virus such as EBV. Recombinanttechnology also can be used to produce antibodies in vitro from clonalpopulations of host cells by transforming the host cells with plasmidscarrying artificial sequences of nucleotides encoding the antibodies.

As used herein, the term “chimeric antigen receptor” or “CAR” as usedherein refers to an antigen-binding domain that is fused to anintracellular signaling domain (also called cytoplasmic signalingdomain) capable of activating or stimulating an immune cell. Mostcommonly, the CAR's extracellular binding domain is composed of a singlechain variable fragment (scFv) derived from fusing the variable heavyand light regions of a murine or humanized monoclonal antibody.Alternatively, scFvs can be used that are derived from Fab's (instead offrom an antibody, e.g., obtained from Fab libraries). In variousembodiments, this scFv is fused to a transmembrane domain and then to anintracellular signaling domain. “First-generation” CARs include thosethat solely provide CD3 signals upon antigen binding,“Second-generation” CARs include those that provide both costimulation(e.g., CD28 or CD137) and activation (CD3). “Third-generation” CARsinclude those that provide multiple costimulation (e.g., CD28 andCD137/4-1BBL) and activation (CD3). In various embodiments, the CAR isselected to have high affinity or avidity for the antigen.

As used herein, a “CD28 polypeptide” refers to a protein having at least85, 90, 95, 96, 97, 98, 99 or 100% identity to GenBank Accession No:NP_001003087.2 or a fragment thereof that has stimulatory activity, forexample, amino acids 115 through 221 of GenBank Accession No:NP_001003087.2. An exemplary CD28 polypeptide is provided below as SEQID NO: 14.

As used herein, a “CD28 nucleic acid molecule” refers to polynucleotideencoding a CD28 polypeptide. An exemplary CD28 nucleic acid molecule isprovided below as SEQ ID NO: 15.

As used herein, a “CD3 zeta chain polypeptide” or “CD3 polypeptide”refers to a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100%identity to GenBank Accession No: XP 005623027.1 or a fragment thereofthat has stimulatory activity, for example, amino acids 65 through 185of GenBank Accession No: XP 005623027.1. An exemplary CD3t polypeptideis provided below as SEQ ID NO: 16.

As used herein, a “CD3 zeta chain nucleic acid molecule” or “CD3 nucleicacid molecule” refers to a polynucleotide encoding a CD3 polypeptide. Anexemplary CD3 nucleic acid molecule is provided below as SEQ ID NO: 17.

As used herein, a “4-1BBL polypeptide” refers to a protein having atleast 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to GenBankAccession No: XP 005633029.1 or a fragment thereof that that acts as atumor necrosis factor (TNF) ligand. An exemplary 4-1BB is provided belowas SEQ ID NO: 18.

As used herein, a “4-1BBL nucleic acid molecule” refers to apolynucleotide encoding a 4-1BBL polypeptide. An exemplary 4-1BBLnucleic acid molecule is provided below as SEQ ID NO: 19.

As used herein, the phrase “activates an immunoresponsive cell” refersto induction of signal transduction or changes in protein expression inthe cell resulting in initiation of an immune response. For example,when CD3 Chains cluster in response to ligand binding and immunoreceptortyrosine-based inhibition motifs (ITAMs) a signal transduction cascadeis produced. In certain embodiments, when an endogenous TCR or anexogenous CAR binds antigen, a formation of an immunological synapseoccurs that includes clustering of many molecules near the boundreceptor (e.g., CD4, CD8, CD3y, CD36, CD3c, or CD3) This clustering ofmembrane bound signaling molecules allows for ITAM motifs containedwithin the CD3 chains to become phosphorylated. This phosphorylation inturn initiates a T cell activation pathway ultimately activatingtranscription factors, such as NF-κB and AP-1. These transcriptionfactors induce global gene expression of the T cell to increase IL-2production for proliferation and expression of master regulator T cellproteins in order to initiate a T cell mediated immune response.

As used herein, the phrase “stimulates an immunoresponsive cell” refersto a signal that results in a robust and sustained immune response. Invarious embodiments, this occurs after immune cell (e.g., T-cell)activation or concomitantly mediated through receptors including, butnot limited to, CD28, CD137 (4-1BB), OX40, CD40 and ICOS. Without beingbound to a particular theory, receiving multiple stimulatory signals isimportant to mount a robust and long-term T cell mediated immuneresponse. Without receiving these stimulatory signals, T cells quicklybecome inhibited and unresponsive to antigen. While the effects of theseco-stimulatory signals vary and remain partially understood, theygenerally result in modifying gene expression (e.g., increasing ordecreasing) in order to generate long lived, proliferative, andanti-apoptotic T cells that robustly respond to antigen for complete andsustained eradication.

As used herein, the term “affinity” refers to a measure of bindingstrength. Without being bound to theory, affinity depends on thecloseness of stereochemical fit between antibody combining sites andantigen determinants, on the size of the area of contact between them,and on the distribution of charged and hydrophobic groups. Affinity alsoincludes the term “avidity,” which refers to the strength of theantigen-antibody bond after formation of reversible complexes. Methodsfor calculating the affinity of an antibody for an antigen are known inthe art, including use of binding experiments to calculate affinity.Antibody activity in functional assays (e.g., flow cytometry assay) isalso reflective of antibody affinity. Antibodies and affinities can bephenotypically characterized and compared using functional assays (e.g.,flow cytometry assay).

As used herein, the term “immunostimulatory activity” refers toinduction of signal transduction or changes in protein expression in acell (e.g., an activated immunoresponsive cell) resulting in an increasein an immune response. Immunostimulatory activity can includepro-inflammatory activity. Polypeptides known to stimulate or increasean immune response via their binding include, but are not limited to,CD28, OX-40, 4-1BB, and their corresponding ligands, including B7-1,B7-2, OX-40L, and 4-1BBL. Such polypeptides are present in the tumormicroenvironment and activate immune responses to neoplastic cells. Invarious embodiments, promoting, stimulating, or agonizingpro-inflammatory polypeptides and/or their ligands enhances the immuneresponse of the immunoresponsive cell.

As used herein, an “epitope” refers to the portion of a molecule that isbound by an antigen-binding protein or fragment or derivative thereof(e.g., by an antibody). An epitope can comprise noncontiguous portionsof the molecule, for example, in a polypeptide, amino acid residues thatare not contiguous in the polypeptide's primary sequence, but that, inthe context of the polypeptide's tertiary and quaternary structure, arenear enough to each other to be bound by an antigen-binding protein).

As used herein, a “CD20 polypeptide” refers to a protein having at least85, 90, 95, 96, 97, 98, 99 or 100% identity to GenBank Accession NO: XP005633357.1 or a fragment thereof that has activating or stimulatoryactivity, such as a CD20 epitope. As used herein, a “CD20 extracellulardomain” refers to a CD20 polypeptide that represents the extracellularportion of a native CD20 protein. A few exemplary CD20 polypeptidesderived from the CD20 extracellular domains of canine, human and mouseCD20 are provided below as SEQ ID NOs: 1, 2 and 3, respectively.

As used herein, the term “isolated” when referring to a molecule, forexample, an antigen-binding protein or fragment or derivative thereof,is a molecule that by virtue of its origin or source of derivation (1)is not associated with naturally associated components that accompany itin its native state, (2) is substantially free of other molecules fromthe same species (3) is expressed by a cell from a different species, or(4) does not occur in nature without human intervention. In other words,an “isolated antigen-binding protein” or “isolated antibody” is onewhich has been identified and separated and/or recovered from acomponent of its natural environment. Thus, a molecule that ischemically synthesized, or synthesized in a cellular system differentfrom the cell from which it naturally originates, will be “isolated”from its naturally associated components. A molecule also can berendered substantially free of naturally associated components byisolation, using purification techniques well known in the art. Moleculepurity or homogeneity can be assayed by a number of means well known inthe art. For example, the purity of a polypeptide sample can be assayedusing polyacrylamide gel electrophoresis and staining of the gel tovisualize the polypeptide using techniques well known in the art. Forcertain purposes, higher resolution can be provided by using HPLC orother means well known in the art for purification.

As used herein, a “conservative amino acid substitution” is one thatdoes not substantially change the structural characteristics of theparent sequence (e.g., a replacement amino acid should not tend to breaka helix that occurs in the parent sequence, or disrupt other types ofsecondary structure that characterize the parent sequence or arenecessary for its functionality). Examples of art-recognized polypeptidesecondary and tertiary structures are described in Proteins, Structuresand Molecular Principles (Creighton, Ed., W.F1. Freeman and Company, NewYork (1984)); Introduction to Protein Structure (C. Branden and J.Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton etal. Nature, 1991, 354: 105, which are each incorporated herein byreference.

As used herein, the term “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression can include splicing of the mRNA in a eukaryotic cell.The expression level of a gene can be determined by measuring the amountof mRNA or protein in a cell or tissue sample. In one aspect, theexpression level of a gene from one sample can be directly compared tothe expression level of that gene from a control or reference sample. Inanother aspect, the expression level of a gene from one sample can bedirectly compared to the expression level of that gene from the samesample following administration of the compositions disclosed herein.The term “expression” also refers to one or more of the followingevents: (1) production of an RNA template from a DNA sequence (e.g., bytranscription) within a cell; (2) processing of an RNA transcript (e.g.,by splicing, editing, 5′ cap formation, and/or 3′ end formation) withina cell; (3) translation of an RNA sequence into a polypeptide or proteinwithin a cell; (4) post-translational modification of a polypeptide orprotein within a cell; (5) presentation of a polypeptide or protein onthe cell surface; and (6) secretion or presentation or release of apolypeptide or protein from a cell.

As used herein, the term “linker” refers to a functional group (e.g.,chemical or polypeptide) that covalently attaches two or morepolypeptides or nucleic acids so that they are connected to one another.As used herein, a “peptide linker” refers to one or more amino acidsused to couple two proteins together (e.g., to couple VH and VLdomains). An exemplary linker sequence used in the present disclosure isGGGGSGGGGSGGGGS (SEQ ID NO: 10).

As used herein, the terms “therapeutically effective” or “effective”refers to an amount of a peptide or composition provided hereineffective to achieve a desired clinical effect. In some embodiments, theamount depends on the condition of a subject and the specific peptideadministered. An effective amount varies with the nature of thecondition being treated, the length of time that activity is desired,and the age and the condition of the subject, and ultimately isdetermined by the health care provider. In some aspects, an effectiveamount of an antigen-binding protein or fragment thereof according tothe present disclosure is an amount effective to reduce or stop tumorgrowth.

As used herein, the term “immunoresponsive cells” refers to any cellthat plays a role in the immune response. Immune cells are ofhematopoietic origin, and include lymphocytes, such as B cells and Tcells; natural killer cells; myeloid cells, such as monocytes,macrophages, dendritic cells, eosinophils, neutrophils, mast cells,basophils, and granulocytes.

As used herein, the term “lymphocyte” refers to all immature, mature,undifferentiated and differentiated white lymphocyte populationsincluding tissue specific and specialized varieties. It encompasses, byway of non-limiting example, B cells, T cells, NKT cells, and NK cells.In some embodiments, lymphocytes include all B cell lineages includingpre-B cells, progenitor B cells, early pro-B cells, late pro-B cells,large pre-B cells, small pre-B cells, immature B cells, mature B cells,plasma B cells, memory B cells, B-1 cells, B-2 cells and anergic AN1/T3cell populations.

As used herein, the term T-cell includes naïve T cells, CD4+ T cells,CD8+ T cells, memory T cells, activated T cells, anergic T cells,tolerant T cells, chimeric B cells, and antigen-specific T cells.

As used herein, the terms “B cell” or “B cells” refers to, by way ofnon-limiting example, a pre-B cell, progenitor B cell, early pro-B cell,late pro-B cell, large pre-B cell, small pre-B cell, immature B cell,mature B cell, naïve B cells, plasma B cells, activated B cells, anergicB cells, tolerant B cells, chimeric B cells, antigen-specific B cells,memory B cell, B-1 cell, B-2 cells and anergic AN1/T3 cell populations.In some embodiments, the term B cell includes a B cell that expresses animmunoglobulin heavy chain and/or light chain on its cells surface. Insome embodiments, the term B cell includes a B cell that expresses andsecretes an immunoglobulin heavy chain and/or light chain. In someembodiments, the term B cell includes a cell that binds an antigen onits cell-surface. In some embodiments disclosed herein, B cells orAN1/T3 cells are utilized in the processes described. In certainembodiments, such cells are optionally substituted with any animal cellsuitable for expressing, capable of expressing (e.g., inducibleexpression), or capable of being differentiated into a cell suitable forexpressing an antibody including, e.g., a hematopoietic stem cell, anaïve B cell, a B cell, a pre-B cell, a progenitor B cell, an earlyPro-B cell, a late pro-B cell, a large pre-B cell, a small pre-B cell,an immature B cell, a mature B cell, a plasma B cell, a memory B cell, aB-1 cell, a B-2 cell, an anergic B cell, or an anergic AN1/T3 cell.

As used herein, an “adoptive cell therapeutic composition” refers to anycomposition comprising cells suitable for adoptive cell transfer. Inexemplary embodiments, the adoptive cell therapeutic compositioncomprises a cell type selected from a group consisting of a tumorinfiltrating lymphocyte (TIL), TCR (e.g., a heterologous T-cellreceptor) modified lymphocytes and CAR (i.e., a chimeric antigenreceptor) modified lymphocytes. In another embodiment, the adoptive celltherapeutic composition comprises a cell type selected from a groupconsisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gammaT-cells, regulatory T-cells and peripheral blood mononuclear cells. Inanother embodiment, TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells,delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclearcells form the adoptive cell therapeutic composition. In one embodiment,the adoptive cell therapeutic composition comprises T cells.

As used herein, “tumor-infiltrating lymphocytes” or TILs refer to whiteblood cells that have left the bloodstream and migrated into a tumor.

As used herein, “neoplasia” refers to a disease characterized by thepathological proliferation of a cell or tissue and its subsequentmigration to or invasion of other tissues or organs. Neoplasia growth istypically uncontrolled and progressive, and occurs under conditions thatwould not elicit, or would cause cessation of, multiplication of normalcells. Neoplasias can affect a variety of cell types, tissues, ororgans, including but not limited to an organ selected from the groupconsisting of bladder, bone, brain, breast, cartilage, glia, esophagus,fallopian tube, gallbladder, heart, intestines, kidney, liver, lung,lymph node, nervous tissue, ovaries, pancreas, prostate, skeletalmuscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, urogenital tract, ureter, urethra, uterus, and vagina, or atissue or cell type thereof. Neoplasias include cancers, such assarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasmacells)

As used herein, a “pathogen” refers to a virus, bacteria, fungi,parasite or protozoa capable of causing disease. Exemplary virusesinclude, but are not limited to, Retroviridae (e.g., humanimmunodeficiency viruses, such as HIV-1 (also referred to as HDTV-III,LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronoviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Naira viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.Hepatitis C); Norwalk and related viruses, and astroviruses).

Exemplary bacteria include, but are not limited to, Pasteurella,Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, andSalmonella species. Specific examples of infectious bacteria include butare not limited to, Helicobacter pyloris, Borelia burgdorferi,Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M.avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeriamonocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtherias, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Rickettsia, and Actinomyces israelli.

As used herein, “signal sequence” or “leader sequence” refers to apeptide sequence (5, 10, 15, 20, 25, 30 amino acids long) present at theN-terminus of newly synthesized proteins that directs their entry to thesecretory pathway. Exemplary leader sequences include the CD8 leadersequence set forth in SEQ ID NO: 12 (canine).

As used herein, “specifically binds” refers to a polypeptide or fragmentthereof that recognizes and binds a biological molecule of interest(e.g., a polypeptide), but which does not substantially recognize andbind other molecules in a sample, for example, a biological sample,which naturally includes a polypeptide of the present disclosure. Forexample, in some embodiments antibodies of the present disclosurespecifically bind canine CD20. In this context “specifically binds”means that the antibody recognizes and binds to canine CD20 with greateraffinity than to other, non-specific molecules that are not canine CD20.For example, an antibody raised against an antigen (polypeptide) towhich it binds more efficiently than to a non-specific antigen (e.g., acanine protein that is not related to or homologous to CD20) can bedescribed as specifically binding to the antigen. Binding specificitycan be tested using, for example, an enzyme-linked immunosorbent assay(ELISA), a radioimmunoassay (RIA), or a western blot assay usingmethodology well known in the art.

As used herein, the term “tumor antigen” refers to an antigen (e.g., apolypeptide) that is uniquely or differentially expressed on a tumorcell compared to a normal cell. With reference to the presentdisclosure, a tumor antigen includes any polypeptide expressed by atumor that is capable of activating or inducing an immune response viaan antigen recognizing receptor (e.g., CD20).

As used herein, the term “virus antigen” refers to a polypeptideexpressed by a virus that is capable of inducing an immune response.

As used herein, the terms “comprises”, “comprising”, and are intended tohave the broad meaning ascribed to them in U.S. Patent Law and can mean“includes”, “including” and the like.

As used herein, the terms “patient,” “subject,” “individual,” and thelike are used interchangeably herein, and refer to an animal, typicallya mammal. In a one embodiment, the patient, subject, or individual is amammal. In one embodiment, the patient, subject or individual is in thecanine family, such as domestic dogs, wolves, foxes, jackals, dingoes.

As used herein, the terms “treating” or “treatment” refers to thetreatment of a disease in a subject, such as a human, and includes: (i)inhibiting a disease, i.e., arresting its development; (ii) relieving adisease, i.e., causing regression of the disease; (iii) slowingprogression of the disease; and/or (iv) inhibiting, relieving, orslowing progression of one or more symptoms of the disease. With respectto a cancer, “treating” or “treatment” also encompasses regression of atumor, slowing tumor growth, inhibiting metastasis of a tumor,inhibiting relapse or recurrent cancer and/or maintaining remission.

It is also to be appreciated that the various modes of treatment orprevention of medical diseases and conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved. The treatment can be a continuous prolongedtreatment for a chronic disease or a single, or few time administrationsfor the treatment of an acute condition.

As used herein, the term “therapeutic” refers to a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

Overview

There are various methods that can be employed to raise an antibodyagainst a protein or peptide antigen. Traditional methods of antibodygeneration are based on digests of proteins to produce peptide antigens,which produce mixtures of antibodies that are purified from bleeds ofimmunized animals. Other methods involve the generation of multiplesynthetic linear peptides that are conjugated to carrier protein forimmunization. Monoclonal antibodies can then be subsequently purified ina complex activity-guided affinity chromatography. In all of the abovemethods, the determination of the antigen sequence is rather cumbersomeand in most cases left unknown. Provided herein is an improved methodwhich takes into account the three dimensional protein structure.

In the present disclosure, the structure of the canine CD20extracellular domain was analyzed and used to recreate a threedimensional canine CD20 antigen for immunization. Based on thisanalysis, a canine extracellular CD20 sequence was chemicallysynthesized in high purity, conjugated to a carrier protein, andinoculated in a suitable animal model for production of CD20 specificantibodies. Unlike other strategies, chemical synthesis provided thecyclized extracellular sequence in a pure form with a structure that isclose to the native extracellular conformation of CD20, which in turnprovided an improved anti-CD20 antibody. Accordingly, the methodsprovided herein using the synthetic CD20 peptide epitope produce highaffinity antibodies to anti-CD20. In some embodiments, the syntheticCD20 peptide epitope can also be used as an affinity reagent bycovalently attaching it to a solid surface, such as a bio-bead or aplate. In some embodiments, attachment is through the same chemistrywhich was used for conjugation to carrier protein. This allows fornecessary quality control assays, such as an ELISA.

Once isolated, the high affinity anti-CD20 antibodies or CD20 bindingfragments thereof, e.g., anti-CD20 antibodies or CD20 binding fragmentsthat bind canine CD20 as provided herein, can be employed in diagnosticand therapeutic methods as described in further detail herein. Inaddition, the anti-CD20 antibodies or CD20 binding fragments can bemodified as described herein to generate fusion proteins, includingchimeric antigen receptors for adoptive cell therapy, using the nucleicacid sequence encoding the anti-CD20 antibodies or CD20 bindingfragments thereof. As described herein in the examples, chimeric antigenreceptors were constructed using the heavy and light chain antigenbinding portions of an anti-CD20 antibody generated using the cyclizedcanine CD20 antigen as described herein. As demonstrated herein, T cellsexpressing these chimeric antigen receptors are able to specificallytarget and kill CD20 expressing tumor cells in vitro and in vivo.Accordingly, the anti-CD20 antibodies or CD20 binding fragments thereofare effective for the treatment of CD20 associated cancers, such as Bcell lymphomas and leukemias.

Generation of an Exemplary CD20 antigen for Antibody Production

CD20 is an activated-glycosylated phosphoprotein expressed on thesurface of all B-cells. CD20 is a membrane-spanning four helix proteinwith both C and N termini located on the cytoplasmic side in a mannerprotruding from two of four trans-membrane helices (FIG. 1 ). On theextracellular domain, there are two loops with the smaller loop composedof amino acids starting at cysteine 167 through cysteine 183 (positionsreferring to human CD20). Together these two cysteines unite throughtheir sulfhydryl side chains by forming a sulfur-sulfur bridge thatcloses the ring (FIG. 2 ). The folding and presentation of the smallloop is crucial to recognition by many therapeutic antibodies such asrituximab and its biosimilars.

The present disclosure provides antigen-binding proteins, for example,antibodies, fragments and derivatives thereof, which specifically bindto a CD20 extracellular domain. The extracellular sequences of canine(SEQ ID NO:1), human (SEQ ID NO:2), and mouse (SEQ ID NO:3) CD20 (FIG. 3, Table 1) show a high degree of similarity though not completeidentity. Therefore, anti-CD20 antibodies developed to one species havea likelihood of interspecies overlap in antibody recognition and can beused for comparative studies. In one embodiment, the antigen-bindingprotein of the present disclosure specifically binds to theextracellular domain of canine CD20 (SEQ ID NO: 1).

Unless otherwise indicated, each polypeptide sequence provided hereinhas an amino terminus at the left and a carboxyl terminus at the right.Polypeptide sequences are indicated using standard one-letterabbreviations.

TABLE 1 SEQ Sequence ID NO CD20 canineNITISHFFKMENLNLIKAPMPYVDIHNCDPANPSEKN 1 Extracellular SLSIQYCGS domainhuman NIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNS 2 PSTQYCYS mouseNMTLSHFLKMRSLNFIRAHTPYINIYNCEPANPSEKN 3 SPSTQYCNS

In some embodiments, the antigen-binding proteins are generated byimmunization of subject with a CD20 antigen provided herein. In someembodiments, the CD20 antigen is derived from an extracellular domain ofa CD20 protein, for example, a canine, human or mouse CD20 extracellulardomain. In particular embodiments, the CD20 antigen is derived from thecanine CD20 extracellular domain of SEQ ID NO:2. In particularembodiments, the CD20 antigen comprises a portion of the canine CD20extracellular domain of SEQ ID NO:2.

In some embodiments, the CD20 antigen is canine CD20 cyclic peptidecomprising a CD20 epitope sequence: YVDIHNCDPANPSEKNSLSIQYC (SEQ ID NO:20), representing a portion of the canine CD20 extracellular domain ofSEQ ID NO:2, in particular, the smaller loop of canine CD20extracellular domain and six additional amino acids of the larger loopof canine CD20 extracellular domain. In some embodiments, the CD20antigen is canine CD20 cyclic peptide, wherein the CD20 portion of theantigen consists of the sequence: YVDIHNCDPANPSEKNSLSIQYC (SEQ ID NO:20). In some embodiments, the canine CD20 cyclic peptide comprises thesequence of SEQ ID NO: 20, wherein the cysteine at position 7 of SEQ IDNO: 20 forms a disulfide bond with the cysteine at position 23 of SEQ IDNO: 20.

In exemplary methods, the canine CD20 epitope sequence for the antigenconstruct includes the 17 amino acids of the smaller extracellular loopof canine CD20 and extended six additional amino acids into the largerextracellular loop: YVDIHNCDPANPSEKNSLSIQYC (SEQ ID NO: 20). Thepresence of the ring and formation of a partial secondary structure isintended mimic that of native extracellular loop of CD20. This allowsfor antibody recognition that is conformational as opposed to linear asin the case of the linear 16 amino acid epitope for rituximab(YNCEPANPSEKNSPST (SEQ ID NO: 21)).

In some embodiments, additional design elements are included in the CD20antigen synthesis procedure to facilitate the chemical synthesis of theantigen construct and/or focus immunogenicity on the CD20 epitopesequence rather than the spacer elements. For example, in someembodiments, the N-terminal amino acid in the construct is kept as afree amino group instead of an acetylated N-terminus in order to give anadvantage to immunogenicity towards N-acetylated termini. In someembodiments, a spacer, such as a small neutral peptide sequence, can beinserted on the C terminal side of the CD20 epitope sequence to insureproper separation and presentation by a carrier protein (e.g., keyholelimpet hemocyanin (KLH)). In some embodiments, the spacer sequence iscomposed of a series of glycine and serine residues. In an exemplaryembodiment, the spacer sequence of Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 58)is employed. In some embodiments, the peptide terminates in a lysine tofacilitate linking chemistry to a carrier protein (e.g., KLH) or othermolecules. In some embodiments, the C-terminal lysine is further linkedto a terminal sulfhydryl group to facilitate linking chemistry to acarrier protein (e.g., KLH) or other molecules. FIG. 4 illustrates anexemplary canine CD20 antigen construct prior to attachment of thecarrier protein. In some embodiments, the carrier protein such as KLH isused without attachment of an artificial immunogenic group such as apolyethylene glycol (PEG) or the like. In some embodiments, the carrierprotein such as KLH is used with attachment of an artificial immunogenicgroup such as a polyethylene glycol (PEG) or the like.

The number of sulfhydryl groups present on the CD20 epitope and theirstructural arrangement vis-a-vis the small extracellular loop and itscorrect formation and consequent conformation presents a challenge tochemical synthesis of the antigen. In particular, there are twosulfhydryl groups emanating from the two cysteine side chains atpositions 7 and 23 of SEQ ID NO:20 (i.e. cysteines 167 and 183 of thefull-length CD20), which must form the extracellular loop, and aterminal sulfhydryl, which is used for linking to KLH. Together, thethree sulfhydryl groups have the tendency to scramble at oxidation orcyclization step should they be liberated at the same time and couldform a multitude of cyclic peptides as well as linear polymers connectedthrough S—S bonds. Therefore, in some embodiments, the synthesis stepsand protecting group strategy are planned such that only the twocysteine side chains are released for cyclization in a synchronousmanner with the terminal sulfhydryl protected during cyclizationreaction.

In some embodiments, an additional connecting group is used to connectthe terminal sulfhydryl group to KLH. The additional connecting groupalso provides further flexibility as well as ease of synthesis byoffering a better protecting group control, for example, in order toexecute on-bead transformations prior to release of the full construct.In some embodiments, a lysine with an orthogonal protecting group, suchas Dde (1-(4,4-dimethyl-2,6,-dioxocyclohex-1-ylidene)ethyl, at its sidechain can be removed on-bead without deprotecting the other amino acidsside chains. Once the lysine side chain is released from Dde protectinggroup, an S-trityl mercapto propionic acid can be connected to theliberated 6-amino group resulting in the full construct on-bead.Selective oxidation of cysteines 167 and 183 and final deblocking of thewhole sequence under non-reducing conditions results in generation ofthe crude construct. This crude construction can then be precipitatedand purified to a high level (e.g., 99+%). In some embodiments, thefractions containing the product can then be lyophilized. In someembodiments, the fractions containing the product can then be conjugatedto KLH, for example, KLH preactivated with a cross-linker such asSulfo-SMCC (sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (FIG. 5 ).

In some embodiments, the KLH conjugated CD20 antigen is then used toinoculate a suitable host for antibody production. Generally, antibodiesare produced by immunizing an animal (e.g., a rodent or domesticatedanimal) with the CD20 antigen, obtaining antibody-producing cells fromthe animal (e.g., by harvesting splenocytes from an animal identified asproducing antibodies following inoculation), and fusing theantibody-producing cells with strains of myeloma cells, e.g., tumorcells, to produce hybridomas which are isolated and cultured asmonoclones. The monoclonal hybridomas can either be cultured in vitro orcan be grown as tumors in a host animal. Because each antibody-producingcell produces a single unique antibody, the monoclonal cultures ofhybridomas each produce a homogeneous antibody which may be obtainedeither from the culture medium of hybridoma cultures grown in vitro orfrom the cells, ascitic fluid, or serum of a tumor-bearing host animal.Once a monoclonal hybridoma is identified, the nucleic acid sequencesencoding the heavy chain and light chains of the antibody can be clonedusing standard techniques and further manipulated to produce theantigen-binding proteins provided herein.

CD20 Antigen Binding Sequences

The present disclosure provides antigen-binding proteins, for example,antibodies, fragments and derivatives thereof, which specifically bindto a CD20 extracellular domain. In some embodiments the antigen-bindingproteins specifically bind to a canine, human, or mouse CD20extracellular domain. In some embodiments the antigen-binding proteinsspecifically bind to a CD20 extracellular domain having the sequence ofSEQ ID NO: 1, 2, or 3 as shown in Table 1. In particular embodiments,the antigen-binding proteins specifically bind to a canine CD20extracellular domain. In particular embodiments, the antigen-bindingproteins specifically bind to a canine CD20 extracellular domain of SEQID NO:1. In some embodiments the antigen-binding proteins are generatedby inoculating a suitable host animal with a canine CD20 cyclic peptidehaving the sequence of SEQ ID NO: 20. In some embodiments, the canineCD20 cyclic peptide comprises the sequence of SEQ ID NO: 20, wherein thecysteine at position 7 of SEQ ID NO: 20 forms a disulfide bond with thecysteine at position 23 of SEQ ID NO: 20. In some embodiments theantigen-binding proteins bind with higher affinity to a CD20extracellular domain having the sequence of SEQ ID NO: 1 as compared toan anti-CD20 antibody generated by using a linear CD20 peptide antigen.

In certain embodiments, the present disclosure provides antigen-bindingproteins, for example, antibodies, fragments and derivatives thereof andfusion proteins comprising amino acid sequences, or encoded by thenucleic acid sequences described in Table 2. For example, provided areantigen-binding proteins, for example, antibodies, fragments andderivatives thereof and fusion proteins comprising a variable heavychain (VH) of SEQ ID NO: 4 and/or a variable light chain (VL) of SEQ IDNO: 6. In some embodiments, provided is an antibody or an antigenbinding fragment thereof comprising a variable heavy chain (VH) of SEQID NO: 4 or a variable heavy chain (VH) having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 4. In some embodiments, provided is an antibody or an antigenbinding fragment thereof comprising a variable light chain (VL) of SEQID NO: 6 or a variable light chain (VL) having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 6.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) complementaritydetermining region (CDR) 1 of SEQ ID NO: 46 or a VL CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 46; (b) a variable light chain (VL)complementarity determining region (CDR) 2 of SEQ ID NO: 48 or a VL CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 48; and/or (c) a variable lightchain (VL) complementarity determining region (CDR) 3 of SEQ ID NO: 50or a VL CDR3 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 50. In someembodiments, provided is an antibody or antigen binding fragment thereofcomprising a variable light chain (VL) complementarity determiningregion (CDR) 1 of SEQ ID NO: 46 or a VL CDR1 having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 46. In some embodiments, provided is an antibody orantigen binding fragment thereof comprising a variable light chain (VL)complementarity determining region (CDR) 2 of SEQ ID NO: 48 or a VL CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 48. In some embodiments, providedis an antibody or antigen binding fragment thereof comprising a variablelight chain (VL) complementarity determining region (CDR) 3 of SEQ IDNO: 50 or a VL CDR3 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 50.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable heavy chain (VH) complementaritydetermining region (CDR) 1 of SEQ ID NO: 40 or a VH CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 40; (b) a variable heavy chain (VH)complementarity determining region (CDR) 2 of SEQ ID NO: 42 or a VH CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 42; and/or (c) a variable heavychain (VH) complementarity determining region (CDR) 3 consisting of athreonine (T) residue. In some embodiments, provided is an antibody orantigen binding fragment thereof comprising a variable heavy chain (VH)complementarity determining region (CDR) 1 of SEQ ID NO: 40 or a VH CDR1having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 40. In some embodiments, providedis an antibody or antigen binding fragment thereof comprising a variableheavy chain (VH) complementarity determining region (CDR) 2 of SEQ IDNO: 42 or a VH CDR2 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 42. In someembodiments, provided is an antibody or antigen binding fragment thereofcomprising a variable heavy chain (VH) complementarity determiningregion (CDR) 3 consisting of a threonine (T) residue.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 of SEQ ID NO: 46 or a CDR1having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 46; (ii) a CDR2 of SEQ ID NO: 48or a CDR2 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity with SEQ ID NO: 48; and/or (iii) aCDR3 of SEQ ID NO: 50 or a CDR3 having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:50; and (b) a variable heavy chain (VH) of SEQ ID NO: 4 or a variableheavy chain (VH) having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 4.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) of SEQ ID NO: 6 or avariable light chain (VL) having at least 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 6; and(b) a variable heavy chain (VH) comprising (i) a complementaritydetermining region (CDR) 1 of SEQ ID NO: 40 or a CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 40; (ii) a CDR2 of SEQ ID NO: 42 or a CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 42; and/or (ii) a CDR3 consistingof a threonine (T) residue.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 of SEQ ID NO: 46 or a CDR1having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 46; (ii) a CDR2 of SEQ ID NO: 48or a CDR2 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity with SEQ ID NO: 48; and/or (iii) aCDR3 of SEQ ID NO: 50 or a CDR3 having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:50; and (b) a variable heavy chain (VH) comprising (i) a complementaritydetermining region (CDR) 1 of SEQ ID NO: 40 or a CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 40; (ii) a CDR2 of SEQ ID NO: 42 or a CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 42; and/or (ii) a CDR3 consistingof a threonine (T) residue.

In some embodiments, provided are antigen-binding proteins, for example,antibodies, fragments and derivatives thereof and fusion proteinscomprising a variable heavy chain (VH) encoded by the nucleic acid ofSEQ ID NO: 5 and/or a variable light chain (VL) encoded by the nucleicacid of SEQ ID NO:7. In some embodiments, provided is an antibody or anantigen binding fragment thereof comprising a variable heavy chain (VH)encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5. In some embodiments, provided is an antibody or an antigen bindingfragment thereof comprising a variable light chain (VL) encoded by thenucleic acid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 7.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) complementaritydetermining region (CDR) 1 encoded by the nucleic acid of SEQ ID NO: 47or a VL CDR1 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 47; (b) a variable light chain (VL) complementaritydetermining region (CDR) 2 encoded by the nucleic acid of SEQ ID NO: 49or a VL CDR2 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 49; and/or (c) a variable light chain (VL) complementaritydetermining region (CDR) 3 encoded by the nucleic acid of SEQ ID NO: 51or a VL CDR3 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 51. In some embodiments, provided is an antibody or antigenbinding fragment thereof comprising a variable light chain (VL)complementarity determining region (CDR) 1 encoded by the nucleic acidof SEQ ID NO: 47 or a VL CDR1 encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 47. In some embodiments, provided isan antibody or antigen binding fragment thereof comprising a variablelight chain (VL) complementarity determining region (CDR) 2 encoded bythe nucleic acid of SEQ ID NO: 49 or a VL CDR2 encoded by the nucleicacid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with SEQ ID NO: 49. In some embodiments,provided is an antibody or antigen binding fragment thereof comprising avariable light chain (VL) complementarity determining region (CDR) 3encoded by the nucleic acid of SEQ ID NO: 51 or a VL CDR3 encoded by thenucleic acid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 51.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable heavy chain (VH) complementaritydetermining region (CDR) 1 encoded by the nucleic acid of SEQ ID NO: 41or a VH CDR1 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 41; (b) a variable heavy chain (VH) complementaritydetermining region (CDR) 2 encoded by the nucleic acid of SEQ ID NO: 43or a VH CDR2 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 43; and/or (c) a variable heavy chain (VH) complementaritydetermining region (CDR) 3 encoded by the nucleic acid consisting of SEQID NO: 45. In some embodiments, provided is an antibody or antigenbinding fragment thereof comprising a variable heavy chain (VH)complementarity determining region (CDR) 1 encoded by the nucleic acidof SEQ ID NO: 41 or a VH CDR1 encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 41. In some embodiments, provided isan antibody or antigen binding fragment thereof comprising a variableheavy chain (VH) complementarity determining region (CDR) 2 encoded bythe nucleic acid of SEQ ID NO: 43 or a VH CDR2 encoded by the nucleicacid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with SEQ ID NO: 43. In some embodiments,provided is an antibody or antigen binding fragment thereof comprising avariable heavy chain (VH) complementarity determining region (CDR) 3encoded by the nucleic acid consisting of SEQ ID NO: 45.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 encoded by the nucleic acidof SEQ ID NO: 47 or a CDR1 encoded by the nucleic acid having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 47; (ii) a CDR2 encoded by the nucleic acid ofSEQ ID NO: 49 or a CDR2 encoded by the nucleic acid having at least 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 49; and/or (iii) a CDR3 encoded by the nucleicacid of SEQ ID NO: 51 or a CDR3 encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 51; and (b) a variable heavy chain(VH) encoded by the nucleic acid of SEQ ID NO: 5 or a variable heavychain (VH) encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 5.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) encoded by thenucleic acid of SEQ ID NO: 7 or a variable light chain (VL) encoded bythe nucleic acid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 7; and (b) avariable heavy chain (VH) comprising (i) a complementarity determiningregion (CDR) 1 encoded by the nucleic acid of SEQ ID NO: 41 or a CDR1encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:41; (ii) a CDR2 encoded by the nucleic acid of SEQ ID NO: 43 or a CDR2encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:43; and/or (ii) a CDR3 encoded by the nucleic acid consisting of SEQ IDNO: 45.

In some embodiments, provided is an antibody or antigen binding fragmentthereof comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 encoded by the nucleic acidof SEQ ID NO: 47 or a CDR1 encoded by the nucleic acid having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 47; (ii) a CDR2 encoded by the nucleic acid ofSEQ ID NO: 49 or a CDR2 encoded by the nucleic acid having at least 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 49; and/or (iii) a CDR3 encoded by the nucleicacid of SEQ ID NO: 51 or a CDR3 encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 51; and (b) a variable heavy chain(VH) comprising (i) a complementarity determining region (CDR) 1 encodedby the nucleic acid of SEQ ID NO: 41 or a CDR1 encoded by the nucleicacid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with SEQ ID NO: 41; (ii) a CDR2 encoded bythe nucleic acid of SEQ ID NO: 43 or a CDR2 encoded by the nucleic acidhaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 43; and/or (ii) a CDR3 encoded bythe nucleic acid consisting of SEQ ID NO: 45.

In some embodiments, provided is a single chain variable fragment (scFv)comprising a heavy chain variable chain of SEQ ID NO: 4 or a variableheavy chain (VH) having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 4 and/or a lightchain variable chain of SEQ ID NO: 6. In some embodiments, provided is asingle chain variable fragment (scFv) comprising a heavy chain variablechain of SEQ ID NO: 4 and/or a light chain variable chain of SEQ ID NO:6 or a variable light chain (VL) having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 6. In some embodiments, the scFv has an amino acid sequence of SEQID NO: 8, or is encoded by the nucleic acid of SEQ ID NO: 9. In someembodiments, the scFv has an amino acid sequence that has at least 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 8. In some embodiments, the scFv is encoded bythe nucleic acid of SEQ ID NO: 9 or a CDR2 encoded by the nucleic acidhaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 9. In some embodiments, the scFvhas a linker sequence that joins the VH and VL chains. In someembodiments, the linker sequence has an amino acid sequence of SEQ IDNO: 10, or is encoded by the nucleic acid of SEQ ID NO: 11.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) complementarity determiningregion (CDR) 1 of SEQ ID NO: 46 or a VL CDR1 having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 46; (b) a variable light chain (VL) complementaritydetermining region (CDR) 2 of SEQ ID NO: 48 or a VL CDR2 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 48; and/or (c) a variable light chain (VL)complementarity determining region (CDR) 3 of SEQ ID NO: 50 or a VL CDR3having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 50. In some embodiments, providedis a single chain variable fragment (scFv) comprising a variable lightchain (VL) complementarity determining region (CDR) 1 of SEQ ID NO: 46or a VL CDR1 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 46. In someembodiments, provided is a single chain variable fragment (scFv)comprising a variable light chain (VL) complementarity determiningregion (CDR) 2 of SEQ ID NO: 48 or a VL CDR2 having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 48. In some embodiments, provided is a single chainvariable fragment (scFv) comprising a variable light chain (VL)complementarity determining region (CDR) 3 of SEQ ID NO: 50 or a VL CDR3having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 50.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable heavy chain (VH) complementarity determiningregion (CDR) 1 of SEQ ID NO: 40 or a VH CDR1 having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 40; (b) a variable heavy chain (VH) complementaritydetermining region (CDR) 2 of SEQ ID NO: 42 or a VH CDR2 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 42; and/or (c) a variable heavy chain (VH)complementarity determining region (CDR) 3 consisting of a threonine (T)residue. In some embodiments, provided is a single chain variablefragment (scFv) comprising a variable heavy chain (VH) complementaritydetermining region (CDR) 1 of SEQ ID NO: 40 or a VH CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 40. In some embodiments, provided is a singlechain variable fragment (scFv) comprising a variable heavy chain (VH)complementarity determining region (CDR) 2 of SEQ ID NO: 42 or a VH CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 42. In some embodiments, providedis a single chain variable fragment (scFv) comprising a variable heavychain (VH) complementarity determining region (CDR) 3 consisting of athreonine (T) residue.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 of SEQ ID NO: 46 or a CDR1having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 46; (ii) a CDR2 of SEQ ID NO: 48or a CDR2 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity with SEQ ID NO: 48; and/or (iii) aCDR3 of SEQ ID NO: 50 or a CDR3 having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:50; and (b) a variable heavy chain (VH) of SEQ ID NO: 4 or a variableheavy chain (VH) having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 4.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) of SEQ ID NO: 6 or a variablelight chain (VL) having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 6; and (b) avariable heavy chain (VH) comprising (i) a complementarity determiningregion (CDR) 1 of SEQ ID NO: 40 or a CDR1 having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 40; (ii) a CDR2 of SEQ ID NO: 42 or a CDR2 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 42; and/or (ii) a CDR3 consisting of athreonine (T) residue.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 of SEQ ID NO: 46 or a CDR1having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 46; (ii) a CDR2 of SEQ ID NO: 48or a CDR2 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity with SEQ ID NO: 48; and/or (iii) aCDR3 of SEQ ID NO: 50 or a CDR3 having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:50; and (b) a variable heavy chain (VH) comprising (i) a complementaritydetermining region (CDR) 1 of SEQ ID NO: 40 or a CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 40; (ii) a CDR2 of SEQ ID NO: 42 or a CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 42; and/or (ii) a CDR3 consistingof a threonine (T) residue.

In some embodiments, provided is a single chain variable fragment (scFv)comprising a variable heavy chain (VH) encoded by the nucleic acid ofSEQ ID NO: 5 and/or a variable light chain (VL) encoded by the nucleicacid of SEQ ID NO:7. In some embodiments, provided is a single chainvariable fragment (scFv) comprising a variable heavy chain (VH) encodedby the nucleic acid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 5. In someembodiments, provided is a single chain variable fragment (scFv)comprising a variable light chain (VL) encoded by the nucleic acidhaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 7.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) complementarity determiningregion (CDR) 1 encoded by the nucleic acid of SEQ ID NO: 47 or a VL CDR1encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:47; (b) a variable light chain (VL) complementarity determining region(CDR) 2 encoded by the nucleic acid of SEQ ID NO: 49 or a VL CDR2encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:49; and/or (c) a variable light chain (VL) complementarity determiningregion (CDR) 3 encoded by the nucleic acid of SEQ ID NO: 51 or a VL CDR3encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:51. In some embodiments, provided is a single chain variable fragment(scFv) comprising a variable light chain (VL) complementaritydetermining region (CDR) 1 encoded by the nucleic acid of SEQ ID NO: 47or a VL CDR1 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 47. In some embodiments, provided is a single chain variablefragment (scFv) comprising a variable light chain (VL) complementaritydetermining region (CDR) 2 encoded by the nucleic acid of SEQ ID NO: 49or a VL CDR2 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 49. In some embodiments, provided is a single chain variablefragment (scFv) comprising a variable light chain (VL) complementaritydetermining region (CDR) 3 encoded by the nucleic acid of SEQ ID NO: 51or a VL CDR3 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 51.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable heavy chain (VH) complementarity determiningregion (CDR) 1 encoded by the nucleic acid of SEQ ID NO: 41 or a VH CDR1encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:41; (b) a variable heavy chain (VH) complementarity determining region(CDR) 2 encoded by the nucleic acid of SEQ ID NO: 43 or a VH CDR2encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:43; and/or (c) a variable heavy chain (VH) complementarity determiningregion (CDR) 3 encoded by the nucleic acid consisting of SEQ ID NO: 45.In some embodiments, provided is a single chain variable fragment (scFv)comprising a variable heavy chain (VH) complementarity determiningregion (CDR) 1 encoded by the nucleic acid of SEQ ID NO: 41 or a VH CDR1encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:41. In some embodiments, provided is a single chain variable fragment(scFv) comprising a variable heavy chain (VH) complementaritydetermining region (CDR) 2 encoded by the nucleic acid of SEQ ID NO: 43or a VH CDR2 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 43. In some embodiments, provided is a single chain variablefragment (scFv) comprising a variable heavy chain (VH) complementaritydetermining region (CDR) 3 encoded by the nucleic acid consisting of SEQID NO: 45.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 encoded by the nucleic acidof SEQ ID NO: 47 or a CDR1 encoded by the nucleic acid having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 47; (ii) a CDR2 encoded by the nucleic acid ofSEQ ID NO: 49 or a CDR2 encoded by the nucleic acid having at least 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 49; and/or (iii) a CDR3 encoded by the nucleicacid of SEQ ID NO: 51 or a CDR3 encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 51; and (b) a variable heavy chain(VH) encoded by the nucleic acid of SEQ ID NO: 5 or a variable heavychain (VH) encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 5.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) encoded by the nucleic acidof SEQ ID NO: 7 or a variable light chain (VL) encoded by the nucleicacid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with SEQ ID NO: 7; and (b) a variableheavy chain (VH) comprising (i) a complementarity determining region(CDR) 1 encoded by the nucleic acid of SEQ ID NO: 41 or a CDR1 encodedby the nucleic acid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 41; (ii) aCDR2 encoded by the nucleic acid of SEQ ID NO: 43 or a CDR2 encoded bythe nucleic acid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 43; and/or (ii)a CDR3 encoded by the nucleic acid consisting of SEQ ID NO: 45.

In some embodiments, provided is a single chain variable fragment (scFv)comprising (a) a variable light chain (VL) comprising (i) acomplementarity determining region (CDR) 1 encoded by the nucleic acidof SEQ ID NO: 47 or a CDR1 encoded by the nucleic acid having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 47; (ii) a CDR2 encoded by the nucleic acid ofSEQ ID NO: 49 or a CDR2 encoded by the nucleic acid having at least 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 49; and/or (iii) a CDR3 encoded by the nucleicacid of SEQ ID NO: 51 or a CDR3 encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 51; and (b) a variable heavy chain(VH) comprising (i) a complementarity determining region (CDR) 1 encodedby the nucleic acid of SEQ ID NO: 41 or a CDR1 encoded by the nucleicacid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with SEQ ID NO: 41; (ii) a CDR2 encoded bythe nucleic acid of SEQ ID NO: 43 or a CDR2 encoded by the nucleic acidhaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 43; and/or (ii) a CDR3 encoded bythe nucleic acid consisting of SEQ ID NO: 45.

In some embodiments, the antigen-binding protein provided hereinspecifically binds to a CD20 protein or extracellular portion thereof invitro and/or in vivo. In some embodiments, the antigen-binding proteinprovided herein specifically binds to a CD20 protein or extracellularportion thereof expressed on the surface of a cell. In some embodiments,the cell is a B cell. In some embodiments, the cell is a cancer cell. Insome embodiments, the cell is a leukemia or a cancer cell.

TABLE 2 SEQ Sequence ID NO α-K9CD20 aminoEVQLVESGGGLVKPGTSLKLSCVASGFSFSDCWM  4 VH acidSWARQTPGKTMEWIGDIKYDGRATNYAPSLQTRF IISRDNAKSTLYLQMTNVRSEDTATYYCTGNHYGGYTLRFAYWGQGTLVTVSS nucleicgaagtacagcttgtggagtctggaggaggtttggtgaaacctgggacttctctg  5 acidaaactctcttgtgtagcctcgggattcagtttcagtgactgctggatgagctgggctcgccagactcctggaaagaccatggagtggattggagatattaaatatgatggcagggccacaaactatgcaccttcccttcagactcgattcataatttccagagacaatgccaagagtaccctgtacctgcagatgaccaatgtgagatctgaggacacagccacttattattgtactgggaaccactacggaggctataccctccggtttgcttactggggccaaggcactctggtcactgtctcttca α-K9CD20 aminoDVVLTQTPPTLSATIGQSVSISCRSSQSLLHSNGNT  6 VL acidYLHWFLQRPGQSPQLLIYLVSRLESGVPNRFSGSG SGTDFTLKISGVEAEDLGVYYCVQGTHAPPTFGGGGAGTNLELKRA nucleicgatgttgtgctgacccagactccacccactttatoggctaccattggacaatcag  7 acidtctccatctcttgcaggtcaagtcagagtctcttacatagtaatggaaacacctatttacattggttcctacagaggccaggccaatctccacagcttctaatttacttggtttccagactggaatctggggtccccaacaggttcagtggcagtgggtcaggaactgatttcacactcaaaatcagtggagtagaggctgaggatttgggagtttattactgtgttcaaggtacccatgctcctccgacgttcggtggcggcggagctgggaccaacctggagctgaaacgggct α-K9CD20 aminoEVQLVESGGGLVKPGTSLKLSCVASGFSFSDCWM  8 scFv acidSWARQTPGKTMEWIGDIKYDGRATNYAPSLQTRF IISRDNAKSTLYLQMTNVRSEDTATYYCTGNHYGGYTLRFAYWGQGTLVTVSSGGGGSGGGGSGGGG SDVVLTQTPPTLSATIGQSVSISCRSSQSLLHSNGNTYLHWFLQRPGQSPQLLIYLVSRLESGVPNRFSGS GSGTDFTLKISGVEAEDLGVYYCVQGTHAPPTFGGGGAGTNLELKRA nucleicgaagtacagcttgtggagtctggaggaggtttggtgaaacctgggacttctctg  9 acidaaactctcttgtgtagcctcgggattcagtttcagtgactgctggatgagctgggctcgccagactcctggaaagaccatggagtggattggagatattaaatatgatggcagggccacaaactatgcaccttcccttcagactcgattcataatttccagagacaatgccaagagtaccctgtacctgcagatgaccaatgtgagatctgaggacacagccacttattattgtactgggaaccactacggaggctataccctccggtttgcttactggggccaaggcactctggtcactgtctcttcaggtggaggtggatcaggtggaggtggatctggtggaggtggatctgatgttgtgctgacccagactccacccactttatcggctaccattggacaatcagtctccatctcttgcaggtcaagtcagagtctcttacatagtaatggaaacacctatttacattggttcctacagaggccaggccaatctccacagcttctaatttacttggtttccagactggaatctggggtccccaacaggttcagtggcagtgggtcaggaactgatttcacactcaaaatcagtggagtagaggctgaggatttgggagtttattactgtgttcaaggtacccatgctcctccgacgttcggtggcggcggagctgggaccaacctggagctgaaacgggc t scFv Linker aminoGGGGSGGGGSGGGGS 10 acid nucleicggtggaggtggatcaggtggaggtggatctggtggaggtggatct 11 acid α-K9CD20 aminoGFSFSDCW 40 VH CDR1 acid nucleic ggattcagtttcagtgactgctgg 41 acidα-K9CD20 amino IKYDGRAT 42 VH CDR2 acid nucleic attaaatatgatggcagggccaca43 acid α-K9CD20 amino TGNHYGGYTLRFAY 44 VH CDR3 acid nucleicactgggaaccactacggaggctataccctccggtttgcttactg 45 acid α-K9CD20 aminoQSLLHSNGNTY 46 VL CDR1 acid nucleic cagagtctcttacatagtaatggaaacacctat 47acid α-K9CD20 amino LVS 48 VL CDR2 acid nucleic ttggtttcc 49 acidα-K9CD20 amino VQGTHAPPTFGG 50 VL CDR3 acid nucleicgttcaaggtacccatgctcctccgacgttcggtggc 51 acid Spacer GSGGGK 52 sequencePD1 amino PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 53 extracellular acidSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ sequencePGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQ nucleic ccaggatggttcttagactccccagacaggccctggaacccccccaccttctc54 acid cccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcacccaggccagccggccag PD-1 signal amino MQIPQAPWPVVWAVLQLGWR 55peptide and acid PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC extracellularSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ sequencePGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQ nucleic atgcagatcccacaggcgccctggccagtcgtctgggcggtgctacaactgg56 acid gctggcggccaggatggttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcacccaggccagccggccag Signal peptide MQIPQAPWPVVWAVLQLGWR 57PD-1

The antigen-binding proteins, fragments and derivatives thereof, andfusion proteins of the present disclosure also include substantiallyhomologous polypeptides that are 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the peptides described in Table 1.

In some embodiments, the antigen-binding proteins, fragments andderivatives thereof, and fusion proteins of the present disclosure canspecifically bind canine CD20 with a wide range of disassociationconstants (Kd). For example, in some embodiments, antigen-bindingproteins, fragments and derivatives thereof, and fusion proteins of thepresent disclosure can bind canine CD20 with a Kd equal to or less thanabout 10⁻⁷ M, such as, but not limited to, 0.1−9.9×10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³, 10⁻¹⁴, 10⁻¹⁵ or any range orvalue therein, as determined by e.g., surface plasmon resonance orKinexa method. The present disclosure encompasses antibodies that bindcanine CD20 polypeptides with a disassociation constant or Kd that iswithin any one of the ranges that are between each of the individualrecited values.

Antigen-binding proteins according to the present disclosure can beprepared by any of a number of conventional techniques. For example,they can be purified from cells that naturally express them (e.g., anantibody can be purified from a hybridoma that produces it), or producedin recombinant expression systems, using any technique known in the art.See, for example, Monoclonal Antibodies, Hybridoma: A New Dimension inBiological Analyses, Kennet et al. (eds.), Plenum Press, New York, 1980;Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York, 1988.

Any expression system known in the art can be used to make therecombinant polypeptides of the present disclosure. In general, hostcells are transformed with a recombinant expression vector thatcomprises DNA encoding a desired polypeptide. Exemplary host cells thatcan be employed for protein expression include prokaryotes, yeast orhigher eukaryotic cells. Prokaryotes include gram negative or grampositive organisms, for example E. coli or Bacillus bacteria. Highereukaryotic cells include mammalian, insect, and plant cells andestablished cell lines thereof. Examples of suitable mammalian host celllines include, but are not limited to the COS-7 line of monkey kidneycells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO)cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVFEBNA cellline derived from the African green monkey kidney cell line CVI (ATCCCCL 70) (McMahan et al., 1991, EMBO J. 10: 2821). Appropriate cloningand expression vectors for use with bacterial, fungal, yeast, andmammalian cellular hosts are described in the art, e.g., by Pouwels etal., Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985. Thetransformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures.

In some embodiments, the antigen-binding proteins according to thepresent disclosure are purified directly from serum of animals immunizedwith an antigen which the antigen-binding proteins, fragments andderivatives thereof, and fusion proteins of the present disclosurespecifically recognizes. In some embodiments, the antigen-bindingproteins according to the present disclosure are purified directly fromhybridoma cell lines or animals bearing hybridoma cell tumors.

The amino acid sequence of the polypeptides disclosed herein can beverified by any means known in the art, and can be identical to thesequences disclosed herein in Table 2, or can differ from thosesequences at one or more amino acid residues as result of processing.For example, on all or a portion of the substantially homogenouspolypeptides, a C-terminal amino acid from either the light chain or theheavy chain (or relevant single-chain molecule) can be removed, byproteolytic processing or other processing that occurs during culture,for example, processing of C-terminal Lys residues. In some embodiments,more than one C-terminal amino acid residue can be removed, for exampletwo C-terminal amino acids, or three, four or five C-terminal aminoacids. Similarly, in some embodiments, N-terminal amino acids can beremoved, for example, one, two, three, four or five N-terminal aminoacids can be removed.

In some embodiments, the antigen-binding proteins, fragments andderivatives thereof, and fusion proteins of the present disclosureundergo post-translational modifications, for example but not limitedto, a glutamine can be cyclized or converted to pyroglutamic acid;additionally, or alternatively, amino acids can undergo deamidation,isomerization, glycation and/or oxidation. The polypeptides of thepresent disclosure can undergo additional post-translationalmodification, including glycosylation, for example N-linked or O-linkedglycosylation, at sites that are well-known in the art. As describedpreviously, changes can be made in the amino acid sequence of apolypeptide to preclude or minimize such alterations, or to facilitatethem in circumstances where such processing is beneficial.

Antigen-binding polypeptides according to the present disclosure can beprepared, and screened for desired properties, by any of a number ofknown techniques. Certain of the techniques involve isolating a nucleicacid encoding a polypeptide chain (or portion thereof) of anantigen-binding protein of interest, and manipulating the nucleic acidthrough recombinant DNA technology. In some embodiments, the nucleicacid is fused to another nucleic acid of interest, or altered (e.g., bymutagenesis or other conventional techniques) to add, delete, orsubstitute one or more amino acid residues, for example.

Polypeptides of the present disclosure include polypeptides that havebeen modified, for example, to: (1) reduce susceptibility toproteolysis, (2) reduce susceptibility to oxidation, (3) alter bindingaffinity for forming protein complexes, (4) alter binding affinities,and (4) confer or modify other physicochemical or functional properties.Additionally, single or multiple amino acid substitutions (e.g.,conservative amino acid substitutions) made in a sequence described inTable 1 (e.g., in the portion of the polypeptide outside the domain(s)forming intermolecular contacts) are encompassed by the presentdisclosure. Consensus sequences can be used to select amino acidresidues for substitution; those of skill in the art recognize thatadditional amino acid residues can also be substituted.

Antigen-binding proteins (e.g., antibodies, antibody fragments, antibodyderivatives, chimeric antigen receptors, and fusion proteins) of thepresent disclosure can comprise any constant region known in the art.The light chain constant region can be, for example, a kappa- orlambda-type light chain constant region, e.g., a human kappa- orlambda-type light chain constant region. The heavy chain constant regioncan be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-typeheavy chain constant regions, e.g., a human alpha-, delta-, epsilon-,gamma-, or mu-type heavy chain constant region. In one aspect, the lightor heavy chain constant region is a fragment, derivative, variant, ormutant of a naturally occurring constant region.

In one aspect, the antigen-binding protein of the present disclosurecomprises a fragment of an antibody. Such fragments can consist entirelyof antibody-derived sequences or can comprise additional sequences.Fragments can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 20, 50, 60, 70, 80, 90, 100, 150 or 200 amino acids in length.Fragments can also be, for example, at most 1,000, 750, 500, 250, 200,175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11,or 10 amino acids in length. Fragments can also result from proteolytic(or other) processing, which, for example, results in variation in theamino and/or carboxyl terminus of from one to five amino acids from thatpredicted. A fragment can further comprise, at either or both of itsends, one or more additional amino acids, for example, a sequence ofamino acids from a different naturally-occurring protein (e.g., an Fc orleucine zipper domain) or an artificial amino acid sequence (e.g., anartificial linker sequence or a tag protein). Amino and carboxyl-terminiof fragments or analogs can occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Computerized comparison methods can be used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. See, e.g., Bowie et al., 1991, Science 253:164.

Examples of antigen-binding fragments include Fab, F(ab′)2, single chainantibodies such as scFvs, diabodies, triabodies, tetrabodies, and domainantibodies. Other examples of antigen-binding fragments are known in theart, e.g., as provided in Lunde et al., 2002, Biochem. Soc. Trans.30:500-06.

In another aspect, the antigen-binding protein of the present disclosurecomprises a derivative of an antibody. The derivative can comprise anymolecule or substance that imparts a desired property, such as increasedhalf-life in a particular use. Examples of molecules that can be used toform a derivative include, but are not limited to, albumin (e.g., humanserum albumin) and polyethylene glycol (PEG). Albumin-linked andPEGylated derivatives of antibodies can be prepared using techniqueswell known in the art.

In one embodiment, the present disclosure provides an isolatedantigen-binding protein or fragment or derivative thereof, comprisingone or more amino acid sequences selected from the group consisting ofSEQ ID NOs: 4, 6 or 8. In one embodiment, the present disclosureprovides an isolated antigen-binding protein or fragment or derivativethereof, comprising one or more amino acid sequences selected from thegroup encoded by SEQ ID NOs: 5, 7 or 9.

In one aspect, an isolated antigen-binding protein of the presentdisclosure is an antibody. In one aspect, the antibody is a full-lengthantibody, a substantially intact antibody, or an antibody fragment,e.g., a Fab fragment, a F(ab′)2 fragment, or a single chain variablefragment (scFv). In some embodiments, a scFv is formed by linking heavyand light chain variable domain (Fv region) fragments via an amino acidbridge (short peptide linker), resulting in a single polypeptide chain.Such single chain Fvs (scFvs) have been prepared by fusing DNA encodinga peptide linker between DNAs encoding the two variable domainpolypeptides (VL and VH). The resulting polypeptides can fold back onthemselves to form antigen-binding monomers, or they can form multimers(e.g., dimers, trimers, or tetramers), depending on the length of aflexible linker between the two variable domains (Kortt et al., 1997,Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different VL and VH-comprising polypeptides, one can formmultimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

In one embodiment, the present disclosure provides an isolated scFvcomprising a VH and a VL, wherein the VH and VL, respectively, compriseamino acid sequences represented by SEQ ID NOs: 4 and 6. In oneembodiment, the present disclosure provides an isolated scFv comprisinga VH and a VL, wherein the VH and VL, respectively, comprise amino acidsequences encoded by the nucleic acid set forth in SEQ ID NOs: 5 and 7.

In one embodiment, the present disclosure provides an isolated scFvcomprising a VH and a VL linked by an amino acid linker. In oneembodiment, the amino acid linker comprises serine and glycine residues.In one embodiment, the linker comprises the amino acid sequence setforth in SEQ ID NO: 10. In one embodiment, the linker comprises theamino acid sequence encoded by the nucleic acid sequence set forth inSEQ ID NO: 11.

In one embodiment, the present disclosure provides an isolated scFvcomprising a VH and a VL linked by an amino acid linker, wherein the VH,VL and linker, respectively, comprise amino acid sequences representedby SEQ ID NOs: 4, 6, and 10. In one embodiment, the present disclosureprovides an isolated scFv comprising a VH and a VL linked by an aminoacid linker, wherein the VH, VL and linker, respectively, comprise aminoacid sequences encoded by the nucleic acid sequences set forth in SEQ IDNOs: 5, 7, and 11.

In one embodiment, the present disclosure provides an isolated scFvcomprising an amino acid sequence represented by SEQ ID NO: 8. In oneembodiment, the isolated scFv comprises an amino acid sequence encodedby the nucleic acid sequence set forth in SEQ ID NO: 9.

In one aspect, the present disclosure provides a fusion proteincomprising an isolated antigen-binding protein or scFv described herein.In one aspect, the fusion protein is a scFv-Fc fusion protein, animmunoconjugate, or a bispecific antibody. In one aspect, the fusionprotein is a scFv-Fc fusion protein comprising a Fc from human IgG1. Thefusion protein can comprise, for example, a detectable (or labeling)moiety (e.g., a radioactive, colorimetric, antigenic or enzymaticmolecule, a detectable bead (such as a magnetic or electrodense (e.g.,gold) bead), or a molecule that binds to another molecule (e.g., biotinor streptavidin)), a therapeutic or diagnostic moiety (e.g., aradioactive, cytotoxic, or pharmaceutically active moiety), or amolecule that increases the suitability of the antibody for a particularuse (e.g., administration to a subject, such as a human subject, orother in vivo or in vitro uses) such as a nanoparticle or liposome. Inone aspect, the fusion protein comprises a component selected from thegroup consisting of a cytotoxin, a detectable label, a radioisotope, atherapeutic agent, a nanoparticle, a liposome, a binding protein, anoligonucleotide, an enzyme, or an antibody.

Generation of Chimeric Antigen Receptors (CAR)

In some embodiments of the present disclosure, the antigen-bindingprotein or scFv is provided in the form of a T cell receptor (TCR) orchimeric antigen receptor (CAR). CARs are engineered receptors, whichgraft or confer a specificity of interest onto an immune effector cell.For example, CARs can be used to graft the specificity of a monoclonalantibody onto an immune cell, such as a T cell. In some embodiments,transfer of the coding sequence of the CAR is facilitated by nucleicacid vector, such as a retroviral vector.

There are currently three generations of CARs. In some embodiments, theantigen-binding protein or scFv is provided in the form of a “firstgeneration” CAR. “First generation” CARs are typically composed of anextracellular antigen binding domain (e.g., a single-chain variablefragment (scFv)) fused to a transmembrane domain fused tocytoplasmic/intracellular domain of the T cell receptor (TCR) chain. Insome embodiments, the extracellular antigen binding domain of the “Firstgeneration” CAR comprises an antigen-binding protein disclosed herein.In some embodiments, the extracellular antigen binding domain of the“First generation” CAR comprises a scFv disclosed herein. “Firstgeneration” CARs typically encode the intracellular domain from the CD3ζchain, which is the primary transmitter of signals from endogenous TCRs.“First generation” CARs can provide de novo antigen recognition andcause activation of both CD4⁺ and CD8⁺ T cells through their CD3t chainsignaling domain in a single fusion molecule, independent ofHLA-mediated antigen presentation.

In some embodiments, the antigen-binding protein or scFv is provided inthe form of a “second generation” CAR. “Second generation” CARs addintracellular domains from various co-stimulatory molecules (e.g., CD28,4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provideadditional signals to the T cell. “Second generation” CARs comprisethose that provide both co-stimulation (e.g., CD28 or 4-IBB) andactivation (e.g., CD3). In some embodiments, the extracellular antigenbinding domain of the “Second generation” CAR comprises anantigen-binding protein disclosed herein. In some embodiments, theextracellular antigen binding domain of the “Second generation” CARcomprises a scFv disclosed herein.

In some embodiments, the antigen-binding protein or scFv is provided inthe form of a “third generation” CAR. “Third generation” CARs comprisethose that provide multiple co-stimulation (e.g., CD28 and 4-1BB) andactivation (e.g., CD3). In some embodiments, the extracellular antigenbinding domain of the “Third generation” CAR comprises anantigen-binding protein disclosed herein. In some embodiments, theextracellular antigen binding domain of the “Third generation” CARcomprises a scFv disclosed herein.

In accordance with the presently disclosed subject matter, the CARs ofthe engineered immune cells provided herein comprise an extracellularantigen-binding domain, a transmembrane domain and an intracellulardomain.

In various embodiments, the antigen-binding protein can be eitherexogenous or endogenous. In some embodiments, the antigen-bindingprotein can be recombinantly produced by fusing portions ofimmunostimulatory proteins together. In some embodiments, theantigen-binding protein comprises a scFv disclosed herein fused to atleast a portion of one or more immunostimulatory proteins. In variousembodiments, the different immunostimulatory proteins can be from thesame or different animal species, such as human, mouse or canine.

In some embodiments, the CARs disclosed herein are “Third generation”CARs. In some embodiments, the CARs further provide costimulation viaone or more costimulatory molecules (also called costimulatory ligands).In some embodiments, the chimeric antigen receptor comprises one or morecostimulatory ligands. In some embodiments, the CAR comprises 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 or more costimulatory ligands. Without intendingto be bound by theory, it is contemplated that the interaction with atleast one co-stimulatory ligand provides a non-antigen-specific signalimportant for full activation of an immune cell (e.g., T cell).Co-stimulatory ligands include, without limitation, tumor necrosisfactor (TNF) ligands, cytokines (such as IL-2, IL-12, IL-15 or IL21),and immunoglobulin (Ig) superfamily ligands. Tumor necrosis factor (TNF)is a cytokine involved in systemic inflammation and stimulates the acutephase reaction. Its primary role is in the regulation of immune cells.Tumor necrosis factor (TNF) ligands share a number of common features.The majority of the ligands are synthesized as type II transmembraneproteins (extracellular C-terminus) containing a short cytoplasmicsegment and a relatively long extracellular region. TNF ligands include,without limitation, nerve growth factor (NGF), CD40L (CD40L)/CD154,CD137L/4-1BBL, tumor necrosis factor alpha (TNFα), CD134L/OX40L/CD252,CD27L/CD70, Fas ligand (FasL), CD30L/CD153, tumor necrosis factor beta(TNF(3)/lymphotoxin-alpha (LTα), lymphotoxin-beta (LT(3), CD257/Bcell-activating factor (BAFF)/Blys/THANK/Tall-1, glucocorticoid-inducedTNF Receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand(TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a largegroup of cell surface and soluble proteins that are involved in therecognition, binding, or adhesion processes of cells. These proteinsshare structural features with immunoglobulins—they possess animmunoglobulin domain (fold). Immunoglobulin superfamily ligandsinclude, without limitation, CD80 and CD86, both ligands for CD28. Insome embodiments, the costimulatory molecule is 4-1BBL.

In some embodiments, the intracellular signaling domain of the antigenrecognizing receptor is the CD4-chain, CD97, CD11a-CD18, CD2, ICOS,CD27, CD154, CDS, OX40, 4-IBB, CD28 signaling domain, a portion thereof,or combinations thereof. In some embodiments, the antigen recognizingreceptor is a CAR comprising at least a portion of CD28, 4-1BB, and/orCD3-chain (see, e.g., Zhong et al., 2010, Molecular Ther.18(2):413-420), together with an antigen binding portion. In someembodiments, the antigen-binding protein is a CAR described in Kohn etal., 2011, Molecular Ther. 19(3):432-438), optionally where the antigenbinding portion is substituted with amino acid sequence that binds toanother tumor or pathogen antigen. In some embodiments, the antigenbinding portion of the chimeric antigen receptor is selected fromimmunoglobulins, variable regions of immunoglobulins (e.g., variablefragments “Fv”), bivalent variable fragments (Fab), and single chainvariable fragments (scFv), etc.

In some embodiments, the chimeric antigen receptor comprises at least aportion of CD28, 4-1BB, and/or CD3-chain, together with a scFv thatspecifically binds to canine CD20 extracellular domain. In someembodiments, the anti-CD20 scFv comprises the amino acid sequence setforth in SEQ ID NO: 8. In some embodiments, the CD28 costimulatorydomain comprises the amino acid sequence set forth in SEQ ID NO: 14. Insome embodiments, the CD3t chain comprises the amino acid sequence setforth in SEQ ID NO: 16. In some embodiments, the 4-1BBL costimulatorydomain comprises the amino acid sequence set forth in SEQ ID NO: 18.Exemplary amino acid and encoding nucleotide sequences for a canine CD8leader, a canine CD28 costimulatory domain, a canine CD3-chain and acanine 4-1BBL costimulatory domain are provided in Table 3. In someembodiments, the chimeric antigen receptor is a recombinantly producedfusion protein comprising one or more segments of amino acid sequencesselected from the group consisting of SEQ ID NO: 4, 6, 8, 10, 12, 14,16, 18, 22, 40, 42, 46, 48, and 50.

TABLE 3 SEQ Sequence ID NO K9CD8 leader amino MASRVTALLLPLALLLRAAAA 12acid nucleic atggcctctcgggtgaccgccctgctcctgccgctggccctgctgctccgtgcc 13acid gcggggcc K9CD28 amino IEVMYPPPYIGNEKSNGTIIHVKEKHLCPDELFPDSS 14 acidKPFWALVVVGAVLVFYSLLVTVALCAYWIKSKSS RILQSDYMNMTPRRPGPTRRHYQPYAPARDFAAY RSnucleic attgaggtcatgtatccacctccttacattggcaatgagaagagcaatgggacca 15 acidttatccatgtgaaagaaaaacatctttgtccagatgagctgtttcctgattcttctaagccattttgggcactggtggtggttggtgcagtcctagttttctatagcttgctagtaacagtggctctttgtgcctactggataaagagtaagagtagcaggatccttcagagtgactacatgaacatgaccccccggaggccggggcccacccgaaggcactaccaaccctatgccccagcacgcgactttgcagcataccgctcc K9CD3ζ aminoRLRSTRPAAPPGAPRGPGQSPRRSSRLLQELNLRG 16 acidREEYEVLDKRRGLDPEMGGKQRKRNPQEVVYNA LQKDKMAEAYSEIGIKSENQRRRGKGHDGLYQGLSTATKDTYDALHMQALPPR nucleiccggctccgctccaccaggcccgcggctcccccgggcgccccacggggtcca 17 acidggccagagcccccgacggtcttcccgcctcctgcaggagctcaatctgcgaggaagagaggagtacgaggttttggataagagacgcggcctggacccggagatgggaggaaagcagaggaagaggaaccctcaggaggtcgtgtacaatgcactgcagaaagacaagatggcagaggcctacagtgagattgggataaaaagcgagaaccagcgtcggagagggaaggggcatgatggcctttaccaggggctcagcacggccaccaaggacacctatgatgccctccacatgcaggccctgcctcctc gc K9 4-1BBL aminoMRPRSDAAPDPEAPRPPAPPGRACSPLPWALSAA 18 acidMLLLVGTCAACALRAWVVPGPRPPALPALPAPLP DAGARLPDSPQAVFAQLVARDVQLKEGPLRWYSDPGLAGVFLGPGLSYDQHTRELMVVEPGLYYVFL HLKLQRVMSSTGSGSVSAALHLQPLGTEAAALDLTLDLPPPSSEARDSAAGFRGSLLHLDAGQRLRVHL RAEAGAHPAWQLAQGATILGLFRVATKVPTGLPSSWPMDTGPGSPPLDGE nucleicatgcgcccccgcagcgacgccgccccggaccccgaggccccgcggccgcc 19 acidcgcgccccccggccgcgcctgcagcccgctgccctgggcgctgagcgccgcgatgctgctgctcgtcggcacctgcgccgcctgcgcgctccgcgcctgggtggtccccgggccccggccccccgcgctccccgcgctccccgcgcccctgccggacgccggcgcccgcctccccgactccccgcaggccgtgttcgcgcagctggtggcccgagatgtacagctgaaggaaggacccctgcgctggtacagtgacccgggcctggcaggtgtattcctggggccgggcctgagttatgaccagcacactcgggagctgatggtggtggaacccgggctctactatgttttcttgcacctgaagctgcagcgggtaatgtccagcacgggctccggctctgtctctgctgccctgcacctgcagccacttggcaccgaggctgcagccctggacctgaccttggacctgcctccaccatcctcggaggcccgtgactcagcagctggtttccggggcagcctgctgcacctggacgcaggccagcgcctccgtgttcacttgcgagctgaggcaggggcccaccctgcctggcagctggcacaaggtgccacgatcttgggcctcttcagagtggccaccaaagtccccactggactcccctcgtcatggcccatggacacggggcctgggtccccgcccctggatggagaa K9 CD34t aminoMLAGRGARAGGGLPRGWTALCLLSLLPFGFTNTE 22 acidTVITPTTVPTSTEIMSAVSENTSKREAITLTPSGTTTLYSVSQDSSGTTATISETTVHVTSTSEITLTPGTMNSSVQSQTSLAITVSFTPTNFSTSSVTLEPSLLPGNGSDPPYNSTSLVTSPTEYYTSLSPTPSRNDTPSTIKGEIKCSGVKEVKLNQGICLELNETSSCEDFKKDNEEKL TQVLCEKEPAEAGAGVCSLLLAQSEVRPHCLLLVLANKTELFSKLQLLRKHQSDLKKLGIRDFTEQDVGS HQSYSRKTLIALVTSGILLAVLGTTGYFLMNRRSWSPTGE nucleic Atgctggcgggcaggggcgcgcgcgcgggcggcgggctgccgcggggct 23 acidggaccgcgctctgcctgctcagtctgctgccctttgggttcacaaacacagaaaccgtgattactcctaccacagtgccaacctccacagaaataatgtcagctgtttctgagaatacatccaaacgggaagccatcacactaactccttctggaactaccaccctgtactctgtctctcaagacagcagtgggaccacagcaaccatctcagagactacagtccatgtcacatctacctctgagatcaccctaacgcctgggaccatgaactcttctgttcagtcgcagacctctttagctatcacggtatcttttaccccaaccaacttttcaacttcaagtgtgaccttggagcccagcctgctacctggaaatggttcggatcccccctacaacagcaccagccttgtgacatcccccacggaatattatacatcactttctcctaccccaagtagaaatgacaccccaagtaccatcaagggagaaatcaaatgttccggagtcaaagaagtgaaattgaaccaaggtatctgcctagagctaaatgagacctccagctgtgaggactttaagaaagataacgaagaaaaactgacccaagtcctgtgtgagaaggagccagctgaggctggggccggggtgtgctccctgcttctggcccagtctgaggtgaggcctcactgcctgctgctggtcttggccaacaaaacagaacttttcagtaaactccaacttctgagaaagcaccagtctgacctgaaaaagctggggatccgagacttcactgaacaagatgttgggagccaccagagctattcccgcaagaccctgattgcactggtcacctcagggatcctgctggctgtcttgggcaccactggttacttcctgatgaaccgccgcagttggagccctaca ggagaa

In some embodiments, provided is a chimeric antigen receptor (CAR)comprising an extracellular antigen binding domain comprising a singlechain variable fragment (scFv), wherein the scFv comprises (a) avariable light chain (VL) complementarity determining region (CDR) 1 ofSEQ ID NO: 46 or a VL CDR1 having at least 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 46;(b) a VL CDR2 of SEQ ID NO: 48 or a VL CDR2 having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 48; and/or (c) a VL CDR3 of SEQ ID NO: 50 or a VL CDR3having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 50. In some embodiments, providedis a CAR comprising an extracellular antigen binding domain comprising ascFv, wherein the scFv comprises a VL CDR1 of SEQ ID NO: 46 or a VL CDR1having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 46. In some embodiments, providedis a CAR comprising an extracellular antigen binding domain comprising ascFv, wherein the scFv comprises a VL CDR2 of SEQ ID NO: 48 or a VL CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 48. In some embodiments, providedis a CAR comprising an extracellular antigen binding domain comprising ascFv, wherein the scFv comprises a VL CDR3 of SEQ ID NO: 50 or a VL CDR3having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 50.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a variable heavy chain (VH) complementarity determining region (CDR) 1of SEQ ID NO: 40 or a VH CDR1 having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:40; (b) a VH CDR2 of SEQ ID NO: 42 or a VH CDR2 having at least 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 42; and/or (c) a VH CDR3 consisting of athreonine (T) residue. In some embodiments, provided is a CAR comprisingan extracellular antigen binding domain comprising a scFv, wherein thescFv comprises a VH CDR1 of SEQ ID NO: 40 or a VH CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 40. In some embodiments, provided is a CARcomprising an extracellular antigen binding domain comprising a scFv,wherein the scFv comprises a VH CDR2 of SEQ ID NO: 42 or a VH CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 42. In some embodiments, providedis a CAR comprising an extracellular antigen binding domain comprising ascFv, wherein the scFv comprises a VH CDR3 consisting of a threonine (T)residue.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a variable light chain (VL) comprising (i) a complementarity determiningregion (CDR) 1 of SEQ ID NO: 46 or a CDR1 having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 46; (ii) a CDR2 of SEQ ID NO: 48 or a CDR2 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 48; and/or (iii) a CDR3 of SEQ ID NO: 50 or aCDR3 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with SEQ ID NO: 50; and (b) a variableheavy chain (VH) of SEQ ID NO: 4 or a variable heavy chain (VH) havingat least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 4.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a VL of SEQ ID NO: 6 or a VL having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:6; and (b) a VH comprising (i) a CDR1 of SEQ ID NO: or a CDR1 having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 40; (ii) a CDR2 of SEQ ID NO: 42 or aCDR2 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with SEQ ID NO: 42; and/or (ii) a CDR3consisting of a threonine (T) residue.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a VL comprising (i) a CDR1 of SEQ ID NO: 46 or a CDR1 having at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 46; (ii) a CDR2 of SEQ ID NO: 48 or a CDR2having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 48; and/or (iii) a CDR3 of SEQ IDNO: 50 or a CDR3 having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 50; and (b) a VHcomprising (i) a CDR1 of SEQ ID NO: or a CDR1 having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 40; (ii) a CDR2 of SEQ ID NO: 42 or a CDR2 having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 42; and/or (ii) a CDR3 consisting of athreonine (T) residue.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises avariable heavy chain (VH) encoded by the nucleic acid of SEQ ID NO: 5and/or a variable light chain (VL) encoded by the nucleic acid of SEQ IDNO:7. In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises aVH encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5. In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises aVL encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:7.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a VL CDR1 encoded by the nucleic acid of SEQ ID NO: 47 or a VL CDR1encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:47; (b) a VL CDR2 encoded by the nucleic acid of SEQ ID NO: 49 or a VLCDR2 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 49; and/or (c) a VL CDR3 encoded by the nucleic acid of SEQ ID NO:51 or a VL CDR3 encoded by the nucleic acid having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 51. In some embodiments, provided is a CAR comprising anextracellular antigen binding domain comprising a scFv, wherein the scFvcomprises a VL CDR1 encoded by the nucleic acid of SEQ ID NO: 47 or a VLCDR1 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 47. In some embodiments, provided is a CAR comprising anextracellular antigen binding domain comprising a scFv, wherein the scFvcomprises a VL CDR2 encoded by the nucleic acid of SEQ ID NO: 49 or a VLCDR2 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 49. In some embodiments, provided is a CAR comprising anextracellular antigen binding domain comprising a scFv, wherein the scFvcomprises a VL CDR3 encoded by the nucleic acid of SEQ ID NO: 51 or a VLCDR3 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 51.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a VH CDR1 encoded by the nucleic acid of SEQ ID NO: 41 or a VH CDR1encoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:41; (b) a VH CDR2 encoded by the nucleic acid of SEQ ID NO: 43 or a VHCDR2 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 43; and/or (c) a VH CDR3 encoded by the nucleic acid consisting ofSEQ ID NO: 45. In some embodiments, provided is a CAR comprising anextracellular antigen binding domain comprising a scFv, wherein the scFvcomprises a VH CDR1 encoded by the nucleic acid of SEQ ID NO: 41 or a VHCDR1 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 41. In some embodiments, provided is a CAR comprising anextracellular antigen binding domain comprising a scFv, wherein the scFvcomprises a VH CDR2 encoded by the nucleic acid of SEQ ID NO: 43 or a VHCDR2 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 43. In some embodiments, provided is a CAR comprising anextracellular antigen binding domain comprising a scFv, wherein the scFvcomprises a VH CDR3 encoded by the nucleic acid consisting of SEQ ID NO:45.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a VL comprising (i) a CDR1 encoded by the nucleic acid of SEQ ID NO: 47or a CDR1 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 47; (ii) a CDR2 encoded by the nucleic acid of SEQ ID NO: 49or a CDR2 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 49; and/or (iii) a CDR3 encoded by the nucleic acid of SEQ IDNO: 51 or a CDR3 encoded by the nucleic acid having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 51; and (b) a VH encoded by the nucleic acid of SEQ IDNO: 5 or a VH encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 5.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a VL encoded by the nucleic acid of SEQ ID NO: 7 or a VL encoded by thenucleic acid having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 7; and (b) a VHcomprising (i) a CDR1 encoded by the nucleic acid of SEQ ID NO: 41 or aCDR1 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 41; (ii) a CDR2 encoded by the nucleic acid of SEQ ID NO: 43 or aCDR2 encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 43; and/or (ii) a CDR3 encoded by the nucleic acid consisting of SEQID NO: 45.

In some embodiments, provided is a CAR comprising an extracellularantigen binding domain comprising a scFv, wherein the scFv comprises (a)a VL comprising (i) a CDR1 encoded by the nucleic acid of SEQ ID NO: 47or a CDR1 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 47; (ii) a CDR2 encoded by the nucleic acid of SEQ ID NO: 49or a CDR2 encoded by the nucleic acid having at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withSEQ ID NO: 49; and/or (iii) a CDR3 encoded by the nucleic acid of SEQ IDNO: 51 or a CDR3 encoded by the nucleic acid having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 51; and (b) a VH comprising (i) a CDR1 encoded by thenucleic acid of SEQ ID NO: 41 or a CDR1 encoded by the nucleic acidhaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 41; (ii) a CDR2 encoded by thenucleic acid of SEQ ID NO: 43 or a CDR2 encoded by the nucleic acidhaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 43; and/or (ii) a CDR3 encoded bythe nucleic acid consisting of SEQ ID NO: 45.

In some embodiments, the CARs disclosed herein further comprise atransmembrane domain. In some embodiments, the transmembrane comprisesat least a portion of the transmembrane domain of the cluster ofdifferentiation (CD) 28 protein. In some embodiments, the CD28 proteinis a human CD28 protein. In some embodiments, the CD28 protein is acanine CD28 protein. In some embodiments, the CAR comprises atransmembrane domain of SEQ ID NO: 14 or a transmembrane domain havingat least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 14. In some embodiments, the CARcomprises a transmembrane domain encoded by the nucleic acid of SEQ IDNO: 15 or a transmembrane domain encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 15. In some embodiments, thetransmembrane domain is fused to the extracellular antigen bindingdomain. In some embodiments, the transmembrane domain is fused to the Cterminus of the extracellular antigen binding domain.

In some embodiments, the CARs disclosed herein further comprise anintracellular signaling domain. In some embodiments, the intracellularsignaling domain comprises at least a portion of the signaling domain ofthe cluster of differentiation (CD) 3ξ protein. In some embodiments, theCD3ξ protein is a human CD3ξ protein. In some embodiments, the CD3protein is a canine CD3ξ protein. In some embodiments, the CAR comprisesan intracellular signaling domain of SEQ ID NO: 16 or an intracellularsignaling domain having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 16. In someembodiments, the CAR comprises an intracellular signaling domain encodedby the nucleic acid of SEQ ID NO: 17 or an intracellular signalingdomain encoded by the nucleic acid having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 17. In some embodiments, the intracellular signaling domain is fusedto the transmembrane domain. In some embodiments, the intracellularsignaling domain is fused to the C terminus of transmembrane domain.

In some embodiments, the CARs disclosed herein further comprise a leadersequence. In some embodiments, the leader sequence comprises at least aportion of the leader sequence of the cluster of differentiation (CD) 8protein. In some embodiments, the CD8 protein is a human CD8 protein. Insome embodiments, the CD8 protein is a canine CD8 protein. In someembodiments, the CAR comprises a leader sequence of SEQ ID NO: 12 or aleader sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with SEQ ID NO: 12. In someembodiments, the CAR comprises a leader sequence encoded by the nucleicacid of SEQ ID NO: 13 or a leader sequence encoded by the nucleic acidhaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 13. In some embodiments, theleader sequence is fused to the extracellular antigen binding domain. Insome embodiments, the leader sequence is fused to the N-terminus ofextracellular antigen binding domain.

In some embodiments, the CARs disclosed herein further comprise asurface marker. In some embodiments, the surface marker comprises atleast a portion of the cluster of differentiation (CD) 34 protein. Insome embodiments, the surface marker comprises a truncated portion ofthe CD34 protein (called CD34t). In some embodiments, the CD34 proteinis a human CD34 protein. In some embodiments, the CD34 protein is acanine CD34 protein. In some embodiments, the CAR comprises a surfacemarker of SEQ ID NO: 22 or a surface marker having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith SEQ ID NO: 22. In some embodiments, the CAR comprises a surfacemarker encoded by the nucleic acid of SEQ ID NO: 23 or a surface markerencoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:23. In some embodiments, the surface marker is fused to the leadersequence. In some embodiments, the surface marker is attached to theleader sequence through a linker sequence.

In some embodiments, the CARs disclosed herein are “Third generation”CARs. In some embodiments, the CARs further provide costimulation viaone or more costimulatory molecules (also called costimulatory ligands).In some embodiments, the costimulatory molecule comprises at least aportion of the 4-1BB ligand (4-1BBL). In some embodiments, the 4-1BBL isa human 4-1BBL. In some embodiments, the 4-1BBL is a canine 4-1BBL. Insome embodiments, the CAR comprises a costimulatory molecule of SEQ IDNO: 18 or a costimulatory molecule having at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ IDNO: 18. In some embodiments, the CAR comprises a costimulatory moleculeencoded by the nucleic acid of SEQ ID NO: 19 or a costimulatory moleculeencoded by the nucleic acid having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:19.

In some embodiments, the costimulatory molecule comprises at least aportion of the programmed cell death protein 1 dominant negativereceptor (PD1-DNR). In some embodiments, the PD1-DNR is a human PD1-DNR.In some embodiments, the PD1-DNR is a canine PD1-DNR. In someembodiments, the PD1-DNR comprises a PD1 signal peptide. In someembodiments, the CAR comprises a costimulatory molecule of SEQ ID NO: 53or a costimulatory molecule having at least 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:53. In some embodiments, the costimulatory molecule further comprises asignal peptide of SE ID NO: 57 or a signal peptide having at least 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with SEQ ID NO: 53. In some embodiments, the CAR comprises acostimulatory molecule of SEQ ID NO: 55 or a costimulatory moleculehaving at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with SEQ ID NO: 55. In some embodiments, the CARcomprises a costimulatory molecule encoded by the nucleic acid of SEQ IDNO: 54 or a costimulatory molecule encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 54. In some embodiments, the CARcomprises a costimulatory molecule encoded by the nucleic acid of SEQ IDNO: 56 or a costimulatory molecule encoded by the nucleic acid having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO: 56.

Nucleic Acids

Genetic modification of immunoresponsive cells (e.g., T cells, CTLcells, NK cells) can be accomplished by transducing a substantiallyhomogeneous cell composition with a recombinant DNA construct.

In one aspect, the present disclosure provides a nucleic acid encodingthe antigen-binding protein or fragment or derivative thereof, isolatedscFv, or a fusion protein described herein. In one embodiment, thenucleic acid encodes an antigen-binding peptide comprising one or moreof amino acid sequences selected from the group consisting of SEQ ID NO:4, 6, 8, 10, 12, 14, 16 and 18. In one embodiment, the nucleic acidcomprises one or more coding sequences operably linked with one another.In some embodiments, the one or more coding sequences are selected fromthe group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17 and 19.

In one embodiment, the nucleic acid encodes a chimeric antigen receptor(CAR) comprising an anti-CD20 scFv and one or more costimulatory domainsselected from a CD28 extracellular signaling domain, a CD3ζ domain ofT-cell receptor (TCR), and a 4-1BBL costimulatory domain. In someembodiments, the anti-CD20 scFv comprises the amino acid sequence setforth in SEQ ID NO: 8. In some embodiments, the CD28 extracellularsignaling domain comprises the amino acid sequence set forth in SEQ IDNO: 14. In some embodiments, the CD3t domain comprises the amino acidsequence set forth in SEQ ID NO: 16. In some embodiments, the 4-1BBLcostimulatory domain comprises the amino acid sequence set forth in SEQID NO: 18.

In one embodiment, the nucleic acid encodes a chimeric antigen receptor(CAR) comprising an anti-CD20 scFv and one or more costimulatory domainsselected from a CD28 extracellular signaling domain, a CD3ζ domain ofT-cell receptor (TCR), and a 4-1BBL costimulatory domain. In someembodiments, the nucleic acid comprises the coding sequence for ananti-CD20 scFv as set forth in SEQ ID NO: 9. In some embodiments, thenucleic acid comprises the coding sequence for the CD28 extracellularsignaling domain as set forth in SEQ ID NO: 15. In some embodiments, thenucleic acid comprises the coding sequence for the CD3t domain as setforth in SEQ ID NO: 17. In some embodiments, the nucleic acid comprisesthe coding sequence for the 4-1BBL costimulatory domain as set forth inSEQ ID NO: 19.

In another related aspect, the present disclosure also provides anexpression vector comprising a nucleic acid described herein, and a hostcell transfected with an expression vector described herein.

In one embodiment, a retroviral vector (either gamma-retroviral orlentiviral) is employed for the introduction of the DNA construct intothe cell. For example, a polynucleotide encoding a chimeric antigenreceptor that binds an antigen (e.g., a tumor antigen, or a variant, ora fragment thereof), can be cloned into a retroviral vector andexpression can be driven from its endogenous promoter, from theretroviral long terminal repeat, or from a promoter specific for atarget cell type of interest. Non-viral vectors, such as CRISPR/Cas, canbe used as well.

For initial genetic modification of the cells, to provide tumor or viralantigen-specific cells, a retroviral vector is generally employed fortransduction, however any other suitable viral vector or non-viraldelivery system can be used. For subsequent genetic modification of thecells, to provide cells expressing an antigen presenting complexcomprising at least two co-stimulatory ligands, retroviral gene transferlikewise proves effective for transduction, however any other suitableviral vector or non-viral delivery system can be used. Combinations ofretroviral vector and an appropriate packaging line are also suitable,where the capsid proteins will be functional for infecting human cells.Various amphotropic virus-producing cell lines are known, including, butnot limited to, PA12 (Miller, et al. (1985)Mol. Cell. Biol. 5:431-437);PA317 (Miller, et al. (1986)Mol. Cell. Biol. 6:2895-2902); HEK293; andCRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464).Non-amphotropic particles are suitable too, e.g., particles pseudotypedwith VSVG, RD114 or GALV envelope and any other known in the art.

Possible methods of transduction also include direct co-culture of thecells with producer cells, e.g., by the method of Bregni, et al. (1992)Blood 80:1418-1422, or culturing with viral supernatant alone orconcentrated vector stocks with or without appropriate growth factorsand polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat.22:223-230; and Hughes, et al. (1992) J Clin. Invest. 89:1817.

Other transducing viral vectors can be used to express a co-stimulatoryligand of the present disclosure in an immunoresponsive cell.Preferably, the chosen vector exhibits high efficiency of infection andstable integration and expression (see, e.g., Cayouette et al., HumanGene Therapy 8:423-430, 1997; Kido et al., Current Eye Research15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649,1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al.,Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors thatcan be used include, for example, adenoviral, lentiviral, andadeno-associated viral vectors, vaccinia virus, a bovine papillomavirus, or a herpes virus, such as Epstein-Barr Virus (also see, forexample, the vectors of Miller, Human Gene Therapy 15-14, 1990;Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al.,Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson,Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller etal., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviralvectors are particularly well developed and have been used in clinicalsettings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson etal., U.S. Pat. No. 5,399,346).

Non-viral approaches can also be employed for the expression of aprotein in a cell. For example, a nucleic acid molecule can beintroduced into a cell by administering the nucleic acid in the presenceof lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am.J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247:1465, 1990). Othernon-viral means for gene transfer include transfection in vitro usingcalcium phosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of asubject can also be accomplished by transferring a normal nucleic acidinto a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue or are injectedsystemically. Recombinant receptors can also be derived or obtainedusing transposases or targeted nucleases (e.g., Zinc finger nucleases,meganucleases, CRISPR nucleases, or TALE nucleases). Transientexpression can be obtained by RNA electroporation.

cDNA expression for use in polynucleotide therapy methods can bedirected from any suitable promoter (e.g., the human cytomegalovirus(CMV), simian virus 40 (SV40), or metallothionein promoters), andregulated by any appropriate mammalian regulatory element or intron(e.g., the elongation factor 1a enhancer/promoter/intron structure). Forexample, if desired, enhancers known to preferentially direct geneexpression in specific cell types can be used to direct the expressionof a nucleic acid. The enhancers used can include, without limitation,those that are characterized as tissue- or cell-specific enhancers.Alternatively, if a genomic clone is used as a therapeutic construct,regulation can be mediated by the cognate regulatory sequences or, ifdesired, by regulatory sequences derived from a heterologous source,including any of the promoters or regulatory elements described above.

The resulting cells can be grown under conditions similar to those forunmodified cells, whereby the modified cells can be expanded and usedfor a variety of purposes.

Immunoresponsive Cells

In some embodiments, the present disclosure provides engineeredimmunoresponsive cells that express a desired chimeric antigen receptor(CAR) or T cell receptor (TCR). Binding of the chimeric antigen receptorto a target antigen activates the immunoresponsive cells to provide anenhanced immune response. In some embodiments, the target antigen is atumor antigen or pathogenic antigen.

Immunoresponsive cells can be obtained using any suitable method knownin the art. In some embodiments, the immune cells are primary immunecells. In some embodiments, the immune cells are lymphocytes, such as Tand B cells. In some embodiments, the immune cells are natural killer(NK) cells. In some embodiments, the immune cells are a mixture oflymphocytes and NK cells. In some embodiments, the immune cells areperipheral blood mononuclear cells (PBMC). In some embodiments, theimmune cells are T cells that have infiltrated a tumor (e.g., tumorinfiltrating lymphocytes). In some embodiments, the T cells are removedduring surgery of a tumor. For example, in some embodiments, the T cellsare isolated after removal of tumor tissue by biopsy. In someembodiments, the immune cells are modified following isolation from adonor.

The unpurified source of immunoresponsive cells can be any known in theart, such as the bone marrow, fetal, neonate or adult or otherhematopoietic cell source, e.g., fetal liver, leukapheresis, peripheralblood or umbilical cord blood. Various techniques can be employed toseparate the cells. For instance, negative selection methods can removenon-T lymphocytes or non-cytotoxic T lymphocytes (CTLs) initially.Monoclonal antibodies (mAbs) are particularly useful for identifyingmarkers associated with particular cell lineages and/or stages ofdifferentiation for both positive and negative selections.

A large proportion of terminally differentiated cells can be initiallyremoved by a relatively crude separation. For example, magnetic beadseparations can be used initially to remove large numbers of irrelevantcells. Preferably, at least about 80%, usually at least 70% of the totalhematopoietic cells will be removed prior to cell isolation.

Procedures for separation include, but are not limited to, densitygradient centrifugation; resetting; coupling to particles that modifycell density; magnetic separation with antibody-coated magnetic beads;affinity chromatography; cytotoxic agents joined to or used inconjunction with a mAb, including, but not limited to, complement andcytotoxins; and panning with antibody attached to a solid matrix, e.g.,plate, chip, elutriation, fluidic method, or any other convenienttechnique.

Techniques for separation and analysis include, but are not limited to,flow cytometry, which can have varying degrees of sophistication, e.g.,a plurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels.

The cells can be selected against dead cells, by employing dyesassociated with dead cells such as propidium iodide (PI). Preferably,the cells are collected in a medium comprising 2% to 5% human serum, 2%fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any othersuitable, preferably sterile, isotonic medium.

In some embodiments, the isolated immunoresponsive cells are geneticallymodified to target tumors or cancer cells through the introduction ofvectors encoding a chimeric antigen receptor as described herein.

Methods of Treatment

In one aspect, the present disclosure provides methods for treating aB-cell cancer in a subject comprising administration of the CD20 antigenbinding proteins or antigen binding fragments thereof or cellsexpressing the CD20 antigen binding proteins or antigen bindingfragments thereof provided herein. Also contemplated herein are methodsfor treating autoimmune diseases comprising administration of the CD20antigen binding proteins or antigen binding fragments thereof or cellsexpressing the CD20 antigen binding proteins or antigen bindingfragments thereof provided herein. Also contemplated herein are methodsfor treating a pathogen infection or other infectious disease in asubject, such as an immunocompromised subject comprising administrationof the CD20 antigen binding proteins or antigen binding fragmentsthereof or cells expressing the CD20 antigen binding proteins or antigenbinding fragments thereof provided herein.

In some embodiments, an effective amount of the antigen-binding proteinprovided herein, or a fragment or analog thereof, are administered toalleviate one or more symptoms of a cancer, an autoimmune disease or aninfectious disease. In some embodiments, the antigen-binding proteinbinds specifically to the extracellular domain of canine CD20 protein.In various embodiments, the antigen-binding protein is a fullimmunoglobulin, an antibody binding fragment (Fab) thereof, the variableregions thereof (Fv) or a recombinantly produced single chain variablefragment (scFv) thereof.

In another related aspect, the present method of treatment relates tocells comprising the nucleic acids or antigen binding proteins disclosedherein, including recombinant immunoresponsive cells, such as, CAR-Tcells genetically modified to express a chimeric antigen receptorcomprising an antigen binding region in accordance with the presentdisclosure. In some embodiments, the method of treatment comprisesisolating immunoresponsive cells from a subject or a donor, transfectingthe isolated cells with an expression vector comprising a nucleic acidencoding an isolated antigen-binding protein or fragment or derivativethereof described herein, and administering the transfected ortransduced immunoresponsive cells to the subject.

In some embodiments, the B-cell cancer is a B-cell lymphoma or a B-cellleukemia. In some embodiments, the B-cell cancer is selected from amongB-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia,B-cell lymphoma, non-Hodgkin's lymphoma, or B-cell malignancies or othermalignancies expressing CD20 or a cross reacting protein or peptide.

In some embodiments, the CD20 antigen binding proteins or antigenbinding fragments thereof or cells expressing the CD20 antigen bindingproteins or antigen binding fragments thereof are administered fortreatment of canine CD20+B-cell lymphoma, immune-mediated hemolyticanemia, immune-mediated thrombocytopenia, and systemic lupuserythematosus (SLE) or other disease or disorder associated withaberrant CD20 or B-cell expression (e.g., over-expression of B cells orCD20 or dysfunctional B cells).

In some embodiments, the autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, systemic lupus erythematosus (SLE),Sjogren's syndrome, vasculitis, multiple sclerosis, Graves' disease,idiopathic thrombocytopenia, dermatomyositis, immune mediatedthrombocytopenia, polymyocytosis, pemphigus, immune mediated hemolyticanemia and bullous pemphigoid.

The methods of treatment of the present disclosure comprisingadministration of the CD20 antigen binding proteins or antigen bindingfragments thereof or cells expressing the CD20 antigen binding proteinsor antigen binding fragments thereof provided herein encompassalleviation or prevention of at least one symptom or other aspect of adisorder, or reduction of disease severity as compared to a subject thathas not been administered the CD20 antigen binding proteins or antigenbinding fragments thereof or cells expressing the CD20 antigen bindingproteins or antigen binding fragments thereof provided herein. Anantigen-binding protein or fragment or derivative thereof, scFv, fusionprotein, nucleic acid, expression vector, host cell, cell expressing aCAR, or pharmaceutical composition described herein need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in the art,therapeutic agents can reduce the severity of a given disease state, butneed not abolish every manifestation of the disease to be regarded asuseful. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject as compared to anon-treated subject, is sufficient.

Dosages and the frequency of administration for use in the methods ofthe present disclosure can vary according to such factors as the routeof administration, the particular antigen-binding proteins employed, thenature and severity of the disease to be treated, whether the conditionis acute or chronic, and the size and general condition of the subject.Appropriate dosages can be determined by procedures known in thepertinent art, e.g., in clinical trials that can involve dose escalationstudies. An antigen-binding protein or fragment or derivative thereof,scFv, fusion protein, nucleic acid, expression vector, host cell, cellexpressing a CAR, or pharmaceutical composition of the presentdisclosure can be administered, for example, once or more than once,e.g., at regular intervals over a period of time. In variousembodiments, time interval between administration of doses of theantigen-binding protein or fragment or derivative thereof, scFv, fusionprotein, nucleic acid, expression vector, host cell, cell expressing aCAR, or pharmaceutical composition can be at least one, two, three,four, five, six, or seven days or one, two, three, four, five, six,seven, or eight weeks, or can be at least one, two, three, four, five,six, seven, eight, nine, ten, or eleven months, or at least one, two,three, or four years. In general, the antigen-binding protein orfragment or derivative thereof, scFv, fusion protein, nucleic acid,expression vector, host cell, cell expressing a CAR, or pharmaceuticalcomposition is administered to a subject until the subject manifests amedically relevant degree of improvement over baseline for the chosenindicator or indicators.

In general, the amount of an antigen-binding protein or fragment orderivative thereof, scFv, or fusion protein described herein present ina dose, or produced in situ by an encoding polynucleotide present in adose, ranges from about 0.01 pg to about 10 mg per kg of host. In oneembodiment, cells expressing a CAR, are administered at a dose of1.5×10⁶ to 3.0×10⁶ CAR-expressing cells/kg. Other host cells can also beadministered at a dose of 1.5×10⁶ to 3.0×10⁶ cells/kg. The use of theminimum dosage that is sufficient to provide effective therapy isusually preferred. Patients can generally be monitored for therapeuticor prophylactic effectiveness using assays suitable for the conditionbeing treated or prevented, which assays will be familiar to thosehaving ordinary skill in the art and which are described herein. Themethods disclosed herein can include oral administration of anantigen-binding protein or fragment or derivative thereof, scFv, orfusion protein or delivery by injection of a liquid pharmaceuticalcomposition. A liquid pharmaceutical composition can include, forexample, one or more of the following: a sterile diluent such as waterfor injection, saline solution, sodium chloride, fixed oils that canserve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents;antioxidants; chelating agents; buffers and agents for the adjustment oftonicity such as sodium chloride or dextrose. A parenteral preparationcan be enclosed in ampoules, disposable syringes, single or multipledose bags or vials made of glass or plastic. The use of physiologicalsaline is preferred, and an injectable pharmaceutical composition ispreferably sterile. When administered in a liquid form, suitable dosesizes will vary with the size of the subject, but will typically rangefrom about 1 ml to about 500 ml (comprising from about 0.01 pg to about10 mg per kg) for a 10-60 kg subject. Optimal doses can generally bedetermined using experimental models and/or clinical trials. The optimaldose can depend upon the body mass, body area, weight, or blood volumeof the subject. As described herein, the appropriate dose can alsodepend upon the patient's (e.g., human) condition, that is, stage of thedisease, tumor burden, general health status, as well as age, gender,and weight, and other factors familiar to a person skilled in themedical art.

In particular embodiments of the methods described herein, the subjectis a human or non-human animal. A subject in need of the treatmentsdescribed herein can exhibit symptoms or sequelae of a disease,disorder, or condition described herein or can be at risk of developingthe disease, disorder, or condition. Non-human animals that can betreated include mammals, for example, non-human primates (e.g., monkey,chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils,hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniaturepig), equine, canine, feline, bovine, and other domestic, farm, and zooanimals.

In some embodiments, the antigen-binding protein or fragment orderivative thereof, scFv, fusion protein, nucleic acid, expressionvector, or host cell described herein are administered in combinationwith one or more therapeutic agents. In some embodiments, the CD20antigen binding proteins or antigen binding fragments thereof or cellsexpressing the CD20 antigen binding proteins or antigen bindingfragments thereof provided herein are administered in combination withone or more therapeutic agents.

In some embodiments, the therapeutic agent is an immunosuppressant,anti-cancer agent, an inhibitor of mitogen-activated protein kinase(MAPK) signaling. In some embodiments, the anti-cancer agent is aproapoptotic agent. In some embodiments, the anti-cancer agent isselected from the group comprising gossyphol, genasense, polyphenol E,Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL),5-aza-2′-deoxycytidine, all trans retinoic acid, doxorubicin,vincristine, etoposide, gemcitabine, imatinib (Gleevec®), geldanamycin,17-N-Ally lamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, orPD184352,Taxol™, also referred to as “paclitaxel”, analogs of Taxol™,such as Taxotere™. In some embodiments, the anti-cancer agent isselected from the group comprising Adriamycin, Dactinomycin, Bleomycin,Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat;bendamustin; benzodepa; bicalutamide; bisantrene hydrochloride;bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium;bropirimine; busulfan; cactinomycin; calusterone; caracemide;carbetimer; carboplatin; carmustine; carubicin hydrochloride;carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatolmesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin Il (includingrecombinant interleukin II, or r1L2), interferon a-2a; interferon a-2b;interferon a-n1; interferon a-n3; interferon b-1 a; interferon y-1 b;iproplatin; irinotecan hydrochloride; lameotide acetate; lenalidomide;letrozole; leuprolide acetate; liarozole hydrochloride; lometrexolsodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vimosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zombicin hydrochloride.

Examples of inhibitors of MAPK signaling include, but are not limitedto, U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063,SP600125, BAY 43-9006, wortmannin, or LY294002; Syk inhibitors; mTORinhibitors; and antibodies (e.g., rituxan).

Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising an antigen-binding protein or fragment orderivative thereof, scFv, fusion protein, nucleic acid, expressionvector, or host cell described herein, and a physiologically acceptablediluent, excipient, or carrier. Optionally, the composition additionallycomprises one or more physiologically active agents, for example, asecond inflammation- or immune-inhibiting substance, an anti-angiogenicsubstance, an analgesic substance, etc. In various particularembodiments, the composition comprises one, two, three, four, five, orsix physiologically active agents in addition to an antigen-bindingprotein or fragment or derivative thereof, scFv, fusion protein, nucleicacid, expression vector, or host cell.

In one aspect, a pharmaceutical composition of the present disclosurecomprises an antigen-binding protein or fragment or derivative thereofdescribed herein with one or more substances selected from the groupconsisting of a buffer, an antioxidant such as ascorbic acid, a lowmolecular weight polypeptide (such as those having fewer than 10 aminoacids), a protein, an amino acid, a carbohydrate such as glucose,sucrose or dextrins, a chelating agent such as EDTA, glutathione, astabilizer, and an excipient. Neutral buffered saline or saline mixedwith conspecific serum albumin are examples of appropriate diluents. Inaccordance with appropriate industry standards, preservatives such asbenzyl alcohol can also be added. The composition can be formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents. Suitable components are nontoxic to recipients at the dosagesand concentrations employed. Further examples of components that can beemployed in pharmaceutical formulations are presented in Remington'sPharmaceutical Sciences, 16tA Ed. (1980) and 20tA Ed. (2000), MackPublishing Company, Easton, Pa.

As is understood in the art, pharmaceutical compositions comprising themolecules of the present disclosure are administered to a subject in amanner appropriate to the indication. A pharmaceutical composition ofthe present disclosure comprising an antigen-binding protein or fragmentor derivative thereof, scFv, fusion protein, or cell expressing a CARdescribed herein can be formulated for delivery by any route thatprovides an effective dose of the immunogen. Pharmaceutical compositionscan be administered by any suitable technique, including but not limitedto parenterally, topically, or by inhalation. If injected, thepharmaceutical composition can be administered, for example, viaintraarticular, intravenous, intramuscular, intralesional,intraperitoneal, intratumoral, or subcutaneous routes, by bolusinjection, or continuous infusion. Localized administration, e.g., at asite of disease or injury is contemplated, as are transdermal deliveryand sustained release from implants. Delivery by inhalation includes,for example, nasal or oral inhalation, use of a nebulizer, inhalation ofthe antagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including tablets, capsules, syrups,lozenges or chewing gum; and topical preparations such as lotions, gels,sprays, patches, and ointments.

Kits

In another aspect, the present disclosure provides a kit comprising anantigen-binding protein or fragment or derivative thereof, scFv, fusionprotein, nucleic acid, expression vector, host cell, cell expressing aCAR, or pharmaceutical composition described herein. Kits for use bymedical practitioners include an antigen-binding polypeptide of thepresent disclosure and a label or other instructions for use in treatingany of the conditions discussed herein. Instructions typically describemethods for administration, including methods for determining the properstate of the subject, the proper dosage amount, and the properadministration method, for administering the composition. Instructionscan also include guidance for monitoring the subject over the durationof the treatment time. Kits provided herein also can include devices foradministration of a pharmaceutical composition described herein to asubject. Any of a variety of devices known in the art for administeringmedications, immunogenic compositions, and vaccines can be included inthe kits provided herein. Exemplary devices include, but are not limitedto, a hypodermic needle, an intravenous needle, a catheter, aneedle-less injection device, an inhaler, and a liquid dispenser, suchas an eyedropper. Typically, the device for administering a compositionis compatible with the active components of the kit.

EXAMPLES Example 1. Synthesis of Canine CD20 Antigen

In this example, an exemplary canine CD20 extracellular antigen waschemically synthesized. The CD antigen contains a CD20 epitope sequencethat includes the 17 amino acids of the smaller extracellular loop ofcanine CD20 and six additional amino acids of the larger extracellularloop: YVDIHNCDPANPSEKNSLSIQYC (SEQ ID NO: 20). The two cysteine residuesform a sulfhydryl bond that cyclizes peptide. The presence of the ringand formation of a partial secondary structure of the cyclic peptide isintended mimic that of native extracellular loop of CD20. This allowsfor antibody recognition that is conformational as opposed to linear asin the case of the linear 16 amino acid epitope for rituximab(YNCEPANPSEKNSPST (SEQ ID NO: 21)).

Additional design elements were included in the CD20 antigen synthesisprocedure to facilitate the chemical synthesis of the antigen constructand/or focus immunogenicity on the CD20 epitope sequence rather than thespacer elements. For example, the N-terminal amino acid in the constructwas kept as a free amino group instead of an acetylated N-terminus inorder to give an advantage to immunogenicity towards N-acetylatedtermini. A spacer, Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 58), was inserted onthe C terminal side of the CD20 epitope sequence to insure properseparation and presentation by the carrier protein, Keyhole limpethemocyanin (KLH). The peptide terminates in a lysine that is furtherlinked to a terminal sulfhydryl group to facilitate linking chemistry tothe carrier protein. FIG. 4 illustrates the exemplary canine CD20antigen construct prior to attachment of the carrier protein.

The number of sulfhydryl groups present on the CD20 epitope and theirstructural arrangement vis-a-vis the small extracellular loop and itscorrect formation and consequent conformation presented a challenge tochemical synthesis of the antigen. In particular, there are twosulfhydryl groups emanating from the two cysteine side chains atpositions 7 and 23 of SEQ ID NO:20 (i.e., cysteines 167 and 183 of thefull-length CD20), which must form the extracellular loop, and aterminal sulfhydryl, which is used for linking to KLH. Together, thethree sulfhydryl groups have the tendency to scramble at oxidation orcyclization step should they be liberated at the same time and couldform a multitude of cyclic peptides as well as linear polymers connectedthrough S—S bonds. Therefore, the synthesis steps and protecting groupstrategy was planned such that only the two cysteine side chains arereleased for cyclization in a synchronous manner with the terminalsulfhydryl protected during cyclization reaction.

FIG. 21 illustrates exemplary synthesis steps and protecting groupstrategy. As shown in FIG. 21 , sulfhydryl group protections wereorthogonal here as they permitted the selective removal and oxidation ofonly the pair that was to be cyclized. Some of the amino acid couplingcould be executed using commercially available pairs that properlyfunctionalized for use in an automatic synthesis setting. For example,the Ser-Leu pair (in purple) was introduced as a pre-functionalized andproperly protected dimer which is commercially available.

An additional connecting group was used to connect the terminalsulfhydryl group to KLH. The additional connecting groups also providedfurther flexibility as well as ease of synthesis by offering a betterprotecting group control, for example, in order to execute on-beadtransformations prior to release of the full construct. As shown in FIG.21 , lysine with an orthogonal protecting group such as Dde at its sidechain was removed on-bead without deprotecting the other amino acidsside chains. Once the lysine side chain was released from Dde protectinggroup, an S-trityl mercapto propionic acid was connected to theliberated 6-amino group resulting in the full construct on-bead. Asshown in FIG. 21 , in anticipation of a final unified and globaldeprotecting step under the same trifluoroacetic acid condition, thisattachment is brought in as an acid-labile trityl group whose removalconditions are comparable to all side chain protecting groups in black;tertiobutyl esters (13u) and tertiobutyoxy carbonyl (Boc) protectinggroups, which are much harder to remove than the monomethoxytrityl (Mmt)groups on the side chain sulfhydryl groups of the two cysteines (influorescent green).

Selective oxidation of cysteines 167 and 183 and final deblocking of thewhole sequence under non-reducing conditions resulted in generation ofthe crude construct. As shown in FIG. 22 , the side chain cysteines (influorescent green) were selectively deprotected with low concentrationTFA (1-5%) under oxidizing conditions which led directly to theselective S—S bond formation which consequently cyclized the peptideinto a ring. This would have been a difficult task in homogeneous phase.

As shown in FIG. 23 , once the peptide cyclized selectively, allprotecting groups were removed and the peptide was deblocked (removed)from resin under non-reducing conditions followed by HPLC purificationalso under non-reducing conditions, lyophilization provided the purepeptide which was divided between a part that was conjugated to KLH forinoculation, a smaller part was used as target for ELISA follow upassessments, and the third part was used for affinity purification.

Once the crude construction was precipitated and purified to a highlevel (e.g., 99% or higher) and lyophilized, the fractions containingthe product were then conjugated to KLH preactivated with across-linker, Sulfo-SMCC ((sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate)) (FIG. 5 ).

Example 2. Immunization of Rats with Synthetic Canine CD20 Peptide

Synthetic canine CD20 peptide as generated above was used to immunizerats. Particularly, four rats were each immunized subcutaneously with 50μg of KLH-peptide conjugate (aka CP-29-KLH) emulsified in TiterMax®adjuvant 8 times over the course of 7 months. Then the rats rested for22 weeks before antigen specific memory B cells were stimulated withacute intravenous boost of the KLH-peptide conjugate (no adjuvant). 3days after, the animals were sacrificed and splenocytes were preparedfor generation of hybridoma cells lines for monoclonal antibodyselection.

A pre-immune bleed was taken prior to administering the firstimmunization dose, and production-bleeds were taken according to thefollowing immunization and bleed schedule:

Procedure Day Procedure Day Prime 1 Boost 1 21 Test bleed 1 28 Boost 242 Test bleed 2 49 Boost 3 63 Test bleed 3 70 Boost 4 84 Test bleed 4 98Boost 5 119 Test bleed 5 126 Boost 6 175 Test bleed 6 182 Boost 7 210Test bleed 7 217 Long Rest Fusion 371

Antibody production in the bleeds was monitored using enzyme-linkedimmunosorbent assay (ELISA). After the color-changing assay wascompleted, the optical density (OD) value of each sample was measured asan indicator of antibody titer within that sample, as antibody titercorrelates positively with the OD value.

All bleeds collected from the immunized animals were screened withELISA. Duplicate ELISA assays were performed, and the date aresummarized in Tables 4 and 5, and plotted in FIGS. 6A and 6B,respectively. In FIGS. 6A and 6B, animal 1 is represented by thediamond, animal 2 is represented by the square, animal 3 is representedby the triangle, and animal 4 is represented by the x. As shown by theresults, the immunization scheme triggered antibody production in allimmunized animals.

TABLE 4 Optical density values in ELISA replica I Animal Bleed 1 2 3 4 0−0.001 −0.001 −0.001 −0.001 1 0.57 0.08 0.02 0.43 2 1.13 0.18 0.33 1.003 1.53 0.35 0.74 0.86 4 1.34 0.39 0.74 0.53 5 1.35 0.48 0.76 0.70 6 1.390.18 0.60 0.81

TABLE 5 Optical density values in ELISA replica II Animal Bleed 1 2 3 40 0.001 0.001 0.001 0.001 1 0.81 0.14 0.19 0.70 2 1.51 0.35 0.51 1.35 31.67 0.57 1.01 1.10 4 1.48 0.60 0.98 0.90 5 1.34 0.74 0.86 0.88 6 1.310.30 0.68 1.01

A serial dilution was performed on the last bleed of Rat No. 4, and theresulting diluted samples were screened by ELISA. The results weresummarized in Table 6, and plotted in FIG. 7 . As shown, further dilutedsamples with lower expected antibody titers consistently showed lower ODvalues, thus confirming the positive correlation between antibody titerand the OD value in this assay.

TABLE 6 Optical density values of serial dilutions of Animal 4, 6thbleed after immunization dilutions OD OD OD OD 500 2.298 2.673 2.4082.459667 1000 2.298 2.302 2.252 2.284 5000 1.992 1.868 1.869 1.90966710000 1.607 1.638 1.615 1.62 15000 1.476 1.413 1.36 1.416333 25000 1.1261.028 1.036 1.063333 50000 0.67 0.642 0.61 0.640667

Specificity of the CD20 antibody produced by the immunized animals wasanalyzed by western blot. NIH3T3 cells were transduced with IRES-purovectors encoding a canine CD20 peptide of between 32 to 36 kilo Dalton(kD) (T). Untransduced 3T3 cells were used as a negative control (UT).The cells were lysed and the contents were homogenized before loadingonto a SDS-PAGE gel. A suitable size standard was also included.

Protein electrophoresis and western blotting were performed according tostandard protocols. Particularly, for western blotting, serum of bleedswere diluted 1000 fold before use as the source of primary antibody(anti-CD20), and an anti-rat secondary antibody was used.

The western blot results are shown in FIG. 8 . The upper panel shows theresults for the pre-immune bleeds of the four immunized animals. Asexpected, no specific band was recognized by western blotting, as thepre-immune bleeds were expected to lack anti-canine CD20 activity. Bycomparison, the lower panel shows the results for the 4^(th) productionbleeds from the four immunized animals. All samples show the specific32-36 kD band corresponding to the canine CD20 peptide expressed by thetransduced 3T3 cells, which was missing for all negative controls usinguntransduced 3T3 cells, as expected. These results confirm anti-canineCD20 specificity of the antibody produced by immunization of animalsusing the synthetic canine CD20 peptide.

Example 3: Construction of Hybridoma Cell Lines

An exemplary protocol for producing hybridoma cell lines is described inthis Example. The hybridoma cell lines were generated by fusing myelomacells with splenocytes. The splenocytes were obtained from rats thatwere immunized with CD20 antigen as described in Examples 1 and 2.

Day 1:

Myeloma cells, P3×63Ag8.653, were passaged twice: early in the morningwith HSFM+10% FBS at 0.5×10⁶ viable cells/ml; and at the end of the dayto with HT media at 0.25×10⁶ viable cells/ml.

Day 2:

Before 10 am, the myeloma cells were passaged with HT media to aconcentration of 0.5×10⁶ viable cells/ml.

The spleen was harvested using a standard protocol from rats that wereimmunized with CD20 antigen and placed in a conical tube containing HTmedia. Generally, rats were euthanized with carbon dioxide as per theAmerican Veterinary Medical Association guidelines, the surgical area onthe rats was disinfected, the abdominal cavity was opened, and thespleen was dissected out.

Blood was collected from the rat by cutting open the chest of the ratsuch that the blood pools in the thoracic cavity after the heart is cut.The blood was transferred into a microfuge tube using a 1 ml syringewithout a needle. To maximize recovery of the blood, the lungs wereremoved prior to cutting the heart. Sera was prepared from the bloodusing a standard protocol.

The spleen and HT media were poured into a 60 mm petri dish. Withoutdisrupting the capsule of the spleen, non-splenic tissue was removedfrom the spleen. The spleen was transferred to a new 60 mm petri dish.

A single cell suspension of splenocytes was obtained by employing the“syringe” method, followed by the “slide” method. To perform the“syringe” method, a 10cc syringe was filled with HT media. A 26-gaugeneedle was attached to the syringe. HT media was slowly injected intothe spleen with the bevel side of the needle up, such that the spleenswelled and RBCs leaked out. Once the injection spot of the spleenappears pale, the syringe was removed and the process of slowlyinjecting the HT media into the spleen was repeated throughout thespleen. If the spleen did not become pale/translucent after injectingthe HT media, the syringe was not refilled with the cell/media mixture.After performing the “syringe” method, the “slide” method was performedby pressing the spleen between the frosted areas of two slides until thespleen started breaking into clumps. The bulk of the organ was pushedabove the frosted area and the clumps were dissociated by gently rubbingthem between the frosted areas. The cells were rinsed off the frostedareas into the HT. The “slide” method was repeated until the spleenappeared completely white or did not become paler.

The splenocyte suspension was passed through a single cell filter into a50 ml conical tube to remove aggregates.

A petri dish was rinsed with ˜2 mls of HT media and the media was passedthrough the same single cell filter that the splenocyte suspension waspassed through into the same 50 ml conical tube that the splenocyteswere collected in.

Cells in the 50 ml conical tube were mixed and ˜100 ul of the cellsuspension was used for counting.

The total cell number and viability of the nucleated splenocytes andmyelomas were determined by dye exclusion technique. Generally, thecells were mixed with eosin (final concentration of 1%) in phosphatebuffered saline and immediately counted with a hemocytometer.

Myeloma cells were added to the splenocytes to obtain a final ratio of 5nucleated splenocytes: 1 myeloma (Total #not Viable #). If necessary,multiple tubes could be used to hold the myeloma cells and the multipletubes can be combined later.

For a control sample, 20 mls of the myeloma were diluted with 20 mls ofFusion Recovery Media and incubated until plating.

The cell mixture was centrifuged. Without disturbing the pellet, themedia was aspirated.

Cells were resuspended in 45 ml serum free HSFM. If multiple tubes wereused to centrifuge the myeloma cells, the myeloma cells were combinedinto the tube containing the splenocytes.

If cell aggregates developed as the sera was washed away, the cellaggregates were removed by passing the cell suspension through anothersingle cell filter.

Repeat the centrifugation step three times, resuspension step two times,and removal of cell aggregates step as needed.

During the final centrifugation, the following items (pre-warmed at justabove 37° C.) were placed into the hood:

-   -   large beaker of water    -   a container (i.e. in a destain box) of warm water with a rack        containing:        -   15 ml conical with 500 ul 50% PEG and a 2 ml pipet        -   50 ml conical with 15 ml HSFM and a 25 ml pipet.

After the final centrifugation, without disrupting the cell pellet, themedia was aspirated.

The cell pellet was disrupted by flicking/tapping the bottom of the tube

The cells were warmed up in the 37° C. beaker of water for 1 minute withmild agitation (by tapping the tube against the wall of the beaker).

Taking care not to touch the cells with the pipet, but rather to coatthe walls of the tube, 500 ul of 50% PEG was added to the cells over 45seconds, with gentle and constant agitation in the 37° C. beaker ofwater

The cells/PEG mixture was immediately diluted with 15 ml HSFM in adrop-wise fashion, over 90 seconds (5 ml/30 seconds), and with gentleand constant agitation in the 37° C. beaker of water.

The cells were incubated without agitation for 8 min at RT in 50 mlconical tube styrofoam rack, then 2 min at just above 37° C. (˜40° C.).

The fusion mixture was centrifuged for 4 minute at 450×g, which is justbefore “5” on the Fisher clinical centrifuge (Centrific™, Model #225,Cat #04-978-50).

The fusion was stopped immediately by aspirating as much media aspossible without disturbing the pellet.

The pellet was resuspended by gently tapping the tube 3 to 5 times.

Resuspension of the pellet was continued with a large bore (i.e. 25 ml)pipet and 30 ml of fresh Fusion Recovery Media by pipetting up and downa few times without forcing the cells between the pipet and bottom ofthe tube.

Any remaining clumps were allowed to settle for a few seconds and then“single” cells were transferred into T75 flask(s).

“Single” cells were continued to be recovered from the tube byadditional aliquots of Fusion Recovery Media and added to the flask(s).If the clumps did not break apart easily, each subsequent wash volumewas pipetted more forcefully.

The final suspension of cells should be approximately 2.5 to 3×10⁶pre-fusion viable cells/ml if recovering >8 hours (preferred) orapproximately 4 to 4.5×10⁶ pre-fusion viable cells/ml if recovering for4 to 8 hours.

The cells in the T25 flask(s) were incubate d for 4 to 20 hours in thecell culture incubator.

Day 3:

150 ul/well of 1.3×HAT media was dispensed into 96 well plates and eachwell was numbered.

In a sterile trough, fused cells were diluted with Fusion Recovery Mediato obtain a concentration of 100×10⁶ pre-fusion viablenucleated/lymphoid in 50 ml (0.1×10⁶/50ul).

Without delay, 50 ul of the diluted fusion suspension was dispensed into10 of the 96 well plates pre-filled with HAT media.

The dilution and dispension of fused cells were repeated until thedesired number of plates were made (i.e. 25). If final set of plates wasless than 10, the same cell concentration as maintained by adjusting thevolume of Fusion Recovery Media added to the wells.

50 ul/well of the control sample (consisting of 20 ml of myeloma cellsdiluted with 20 ml of Fusion Recovery Media) was plated into 1pre-filled 96 well plate.

Plates were incubated for 5 to 8 days undisturbed. One or two plates canbe checked for growth, but care should be taken to not disturb colonies.

Days 7 to 10:

To determine fusion efficiency, the number of clones was counted(cluster of ≥10 spherical/glowing cells) using a 4× objective, usuallyon day 8 or 9. The number of colonies/well and percent wells withhybridoma growth were recorded.

The fusion was fed on day 9 or 10 by aspirating the media and adding 150ul Fusion Recovery Media. If the clones were particularly small (mostcolonies are <25 cells) or infrequent (<0.5 clones/well), then feedingbegan on the Day 7 or 8. If the clones were particularly large (e.g.,can be seen by the naked eye on day 7 or 8) and show noticeable growthby day 9 or 10, then feeding was delayed until day 13.

Day 23 to 30:

For Quality Control data, the number hybridomas from all plates wascounted and the percent of wells from all plates that containedhybridomas was calculated. The number of colonies/well was adjusted withthis final count.

Materials used in Example 3:

-   -   Myelomas:        -   P3×63Ag8.653 For rats/hamsters (ATCC/Cat #CRL-1580)    -   Reagents:        -   Gentamycin: (Invitrogen/Cat. #15710-064)        -   50% PEG 1500: in 75 mM Hepes (w/v) (Roche/Cat. #783 641)        -   Hypoxanthine, Thymidine (HT):(100× from Invitrogen/Cat.            #11067-030)        -   Hypoxanthine, Aminopterin, Thymidine (HAT): (50×HAT from            Sigma/Cat. #H0262)    -   Media/Supplements:        -   Hybridoma Serum Free Media (HSFM)            -   (Invitrogen/Cat. #12300-067 . . . for powder)            -   (Invitrogen/Cat. #12045-076 . . . 1000 ml liquid)            -   (Invitrogen/Cat. #12045-084 . . . 500 ml liquid)        -   Fetal Bovine Serum (FBS) (pre-screened fusion/cloning            quality)        -   Growth Factor Supplement Need to be titered for each new lot            -   For rat/hamster fusions Hybridoma Cloning Factor (HCF)    -   Fusion Base Media:        -   HSFM        -   15% FBS        -   1× Appropriate Growth Factor Supplement        -   10 mg/ml Gentamycin    -   HT Media (Fusion Growth Media):        -   Fusion Base Media        -   1×HT    -   Fusion Recovery Media:        -   HT media        -   2× Growth Factor Supplement    -   1.3×HAT Media (Fusion Selection Media=1×):        -   Fusion Base Media        -   2× Growth Factor Supplement rather than 1×        -   1.3×HAT    -   Equipment:        -   Cell Culture Incubator maintained at:            -   37° C.            -   7% CO₂            -   Humidified    -   Misc Items:        -   500 ml FRESH 70% EtOH made in bottle        -   60 mm petri dishes        -   1 ml syringe        -   10 ml syringe and 26 g needle        -   Sterile frosted glass slides for disrupting spleen—Prepared            by scraping the excess frosting off with the edge of another            slide, washing glass dust off the slides with ultra pure            water and autoclaving the slides in sets of 2/bag.        -   Sterile single cell filter (Fisher/Cat #08-771-1)        -   Sterile flat bottom 96 well tissue culture plates

Construction of Cells Expressing the K9CD20 Antigen

Construction of SFG-K9CD20-dsRed Plasmid

The plasmid SFG-K9CD20-dsRed was used to express K9CD20 antigen inNIH3T3, EL4 and NALM-6 cells. It was prepared by replacing the IRES-Puroelements of the SFG-K9CD20-IRES-Puro construct with P2A-dsRed.K9CD20P2AdsRed was generated with overlapping PCR using equal molarmixture of 2 PCR fragments. Fragment 1 consisting of PmlI-K9CD20 and P2Awas generated using primers k9CD20-F1:5′-GGCCCACGTGGCCACCATGACAACACCCAGAAATT-3′ (SEQ ID NO: 30) and K9CD20-R1:5′-GGGTCCGGGATTCTCCACGTCACCTGCTTGTTTGAGTAGTGAGAAGTTTGTTGCTCCAGATCCAGGGATGCTGTCGTTTTCTATTGGT-3′(SEQ ID NO: 31) usingSFG-K9CD20-IRES-Puro. Fragment 2 consisting of P2A C-terminalsequence-dsRed-BamHI site was generated with primersK9CD20-F2:5′—CAAGCAGGTGACGTGGAGGAGAATCCCGGACCCATGGACAACACCGAGGACGTCAT-3′ (SEQ ID NO: 32) andk9CD20-R2:5′-TTAAGGATCCCTACTGGGAGCCGGAGTGGCGGG-3′ (SEQ ID NO: 33) usingSFG-PZ1-IRES-dsRed as template. The primers used for overlapping PCRwere K9CD20-F1 and K9CD20-R2. K9CD20dsRed was cloned into the SFG vectorbackbone between the PmlI and BamHI sites to produce the vectorSFG-K9CD2O-P2A-dsRed. All PCR reactions were performed using ProFlex PCRsystem (Applied Biosystems) and Platinum PCR supermix (Invitrogen) kitper manufacture's recommendations. The resulting cassette K9CD20P2AdsRedwas cloned in the backbone of SFG-anti-K9CD2028zLNGFR by replacing thePmlI-BamH1 cassette as shown in FIG. 14K. FIG. 14D shows an illustrationof the K9CD20dsRed cassette and FIG. 14J an illustration of theSFG-vector backbone.

Example 4: Construction and Characterization of K9CD20-Targeted CAR TCells

In this Example, the methods for generating and analyzing CAR T cellsare described.

Construction of an anti-canine CD20 single-chain variable fragment(scFv)-CD3ξ-chain fusion gene (K9CAR)

To construct an anti-canine CD20-specific scFv, a rat antibody targetedto canine CD20 (K9CD20) was generated by immunization of rats with theCD20 antigen shown in FIG. 5 and as described in Examples 1 and 2.Splenocytes were isolated from the immunized rats and used to generatehybridoma cell lines as described in Example 3. Hybridomas were screenedby ELISA. Hybridomas that were positive by ELISA were further screenedby flow cytometry analysis using NIH3T3 cells expressing k9CD20(3T3-K9CD20). 3T3-K9CD20 cells were stained with the hybridomassupernatant (and with secondary anti-Rat IgG FITC antibody) and analyzedby flow cytometry (FIGS. 17A-B, 18A-B, and 19A-B). As shown in FIGS.17B, 18B, and 19B, respectively, clones 5B3, 10C10 and 18F6 were allpositives for anti-K9CD20 antibody production. As a negative control,clone 7A7 K9 was confirmed negative for anti-K9CD20 antibody (FIG. 20B).

cDNA was isolated from the anti-K9CD20 antibody-producing hybridoma(18F6 clone). The nucleic acid encoding light chain (SEQ ID NO: 4) andheavy chain (SEQ ID NO: 6) variable regions of the rat anti-K9CD20antibody were cloned from the cDNA isolated from the hybridoma 18F6clone by 5′ Rapid Amplification of cDNA Ends (5′RACE) using thefollowing gene specific primers (GSPs):

SEQ ID Sequence NO: GSP (r2a)-1 5′-GGAAATAGCCCTTGACCAGGC 24 GSP (r2a)-25′-GAGCCAGTGGATAGACAGATG 25 GSP (r2a)-3 5′-GTGGATAGACAGATGGGGCTG 26GSP (κ)-1 5′-AGGATGATGTCTTATGAACAA 27 GSP (κ)-25′-ATGAACAACCTCACAGGTATAGAGG 28 GSP (κ)-3 5′-CTCACAGGTATAGAGGTTATG 29The GSP (r2a)-1, GSP (r2a)-2, and GSP (r2a)-3 primers were used to clonethe heavy chain variable region (HCVR) and the GSP (x)-1, GSP 00-2, andGSP 00-3 primers were used to clone the light chain variable region(LCVR). FIG. 14A shows an illustration of the cloning of the light chainand heavy chain variable regions using 5′RACE.

A single-chain fragment variable (scFv) antibody (SEQ ID NO:8) (calledanti-K9CD20 scFv) was generated by fusing the nucleic acids encodinglight and heavy chains separated by nucleic acid encoding a Gly-Serlinker (SEQ ID NO: 10). FIG. 14B shows an illustration of theanti-K9CD20 scFv.

The anti-K9CD20 scFv was further modified by fusing the human CD8 (hCD8)leader sequence, human CD28 (hCD28) transmembrane domain, and theintracellular signaling domain of the CD3 zeta chain of the human T cellreceptor (TCR) (hCD3z) to generate a chimeric antigen receptor (CAR)named K9CAR (or SFG-anti-K9CD20 CAR LNGFR). FIG. 14C shows anillustration of the K9CAR cassette.

Construction of K27

The K27 (also called K27CAR) cassette contains three canine components,the canine CD8 leader (K9CD8; SEQ ID NO:12), canine CD28 transmembranedomain (K9CD28; SEQ ID NO:14), and canine CD3z signaling domain (K9CD3z;SEQ ID NO:16), in addition to the anti-K9CD20scFv sequence. The K27 DNAfragment was generated by replacing the hCD8 leader, hCD28, and hCD3zregions in the K9CAR cassette with the corresponding canine sequences.FIG. 14E shows an illustration of the K27 cassette.

Construction of SGF-K27/41BBL

The K27/41BBL (also named K27CAR/41BBL and K36CAR) cassette contains theK27 cassette and the canine 41BBL costimulatory ligand (SEQ ID NO:18).Sequences of K9CD8leader-anti-K9CD20ScFv-K9CD28-K9CD3zeta-P2A-K9-41BBLflanked by Agel andXhol sites was synthesized in pUC backbone by Blue Heron Technology(pUC-K36). The fragment between Age I and Xho I in pUC-K36 was used toreplace the partial SFG backbone and 1928z sequences between Agel andXho I site of the SFG-1928z vector (described in Hollyman et al., J.Immunother, 32(2):169-80 (2009)) to produce the SFG-K36 vector. FIG. 14Fshows an illustration of the K36CAR cassette.

Construction of SFG-K27

The SFG-K27 vector contains the K27 cassette. The SFG-K27 vector wasproduced by amplifying the K27 cassette using forward primer:5′-GGCCGGATCCTTCAGAGTGACTACATGAA-3′ (SEQ ID NO: 34) and reverse primer:GCGGCCGCTCAGCGAGGAGGCAGGGCCTGCATG-3′ (SEQ ID NO: 35) using thesynthesized pUC-K36 plasmid as template. The K36 DNA fragment in SFG-K36between BamHI sites was replaced by the K27 DNA fragment to produce theSFG-K27 vector. Orientation of K27 insert was confirmed by sequencing.

Construction of SFG-K9CD34t-K2 7 and SFG-k9CD34t-K36

The K9CD34t sequence (SEQ ID NO: 23) was synthesized and cloned in pUCby Blue Heron Technology (pUC-K9CD34t). The K9CD34t DNA fragment with anAfe I restriction site at the 5′ end was amplified was amplified withprimers k9CD34-F1-5′-GGCCAGCGCTGCCACCATGCTGGCGG (SEQ ID NO: 36) andk9CD34-R1-5′-GGGTCC AGGGTTCTCCTCCACGT (SEQ ID NO: 37) using pUC-K9CD34tplasmid as template. The K27 DNA fragment with overlapping sequence withK9-CD34 on the 5′ end and a SbfI restriction site on the 3′ end wasamplified with primersk9CD34-F2-5′—GCTGGAGACGTGGAGGAGAACCCTGGACCCATGGCCTCTCGGGTGACCGCCC (SEQID NO: 38) and k9CD34-R2-5′-TTAACCTGCAGGAGGCGGGAAGACCG (SEQ ID NO: 39)using SFG-K27 as template. The K9CD34t-K27 DNA fragment was amplifiedwith primers k9CD34-F1 and k9CD34-R2 using the equal molar mixture ofthe K9CD34t and K27 PCR products. The K27 element in SFG-K27 wasreplaced by K9CD34t-K27 using the AfeI and SbfI sites to produce theSFG-K9CD34t-K27 vector construct. The K27 element in SFG-K36 wasreplaced by K9CD34t-K27 using the AfeI and SbfI sites to get vectorSFG-k9CD34t-K36. FIGS. 14G and 14H show illustrations of the K9CD34t-K27and K9CD34t-K36 cassettes, respectively.

Human T-Cell Cultures, Activation and Retroviral Transduction.

Blood samples were obtained from a healthy donor. Peripheral bloodmononuclear cells (PBMC) were separated on Ficoll, then activated andmagnet selected with CD3/CD28 dynabeads at 1:1 ratio cultured in X-VIVO15 containing 5% human serum, 2 mmol/L L-glutamine (Life Technologies),100 units/mL penicillin, and 100 ug/mL streptomycin (Life Technologies),100 units/ml of IL2 (R&D Systems), HEPES buffer and pyruvate Na (day 0).After 72 hours (day 3), the human T cells were transduced bycentrifugation on retronectin-coated 6-well plates with retroviralsupernatant (1:1 of volume ratio) at the final density about 0.35E+6cells/ml. On day 7, the cells were analyzed by FACS analysis fortransduction efficiency. At day 10 or after, transgene expression wasmeasured again by FACS analysis, and cytotoxicity assays were performed.

Characterization of CAR T Cells by Flow Cytometry

Flow cytometry was performed on a BD-LSRII cytometer and data analyzedwith FlowJo software (Treestar). The following mAbs were used forphenotypic analysis for human T cells: phycoerythrin (PE)-labeled antihuman LNGFR (CAR), APC-conjugated anti-human CD3, PacificBlue-conjugated anti-human CD4 (BD Biosciences), and PE-Cy7-conjugatedanti-human CD8. The following mAbs were used for phenotypic analysis fortumor cells NALM6: GFP for luciferase and dsRed for canine CD20.

FIG. 10A shows the transduction efficiency of the anti-K9CD20 CARco-expressing the L-NGFR (using the SFG-anti-K9CD20 CAR LNGFR vector asshown in FIG. 14C) in human T cells as measured by flow cytometry. Thetransduction efficiency was assessed by coexpression of L-NGFR. As shownin FIG. 10A, transduced human T cells expressed L-NGFR (top row),whereas non-transduced human T cells did not express L-NGFR (bottomrow), and the transduction efficiency in human T cells was approximately30%.

Cytotoxic T Lymphocyte (CTL) Assays

The cytotoxic activity of human T cells transduced with K9CD20constructs was assessed by standard 51 Cr release assays. Briefly, humanT cells were transduced with the SFG-anti-K9CD20 CAR LNGFR construct,which results in expression of the CAR targeted to K9CD20 together withhu-LNGFRt, hu-CD28 signaling domain and hu-CD3z chain. Transduced Tcells were assessed by LNGFR-PE for CAR expression as well as CD4:CD8ratio on the same performing CTL. NALM-6 or EL4 tumor cells were labeledwith 51 Cr for 1 hour at 37° C., washed with RPMI medium supplementedwith 10% FCS, and resuspended in the same medium at a concentration of1×10⁵ tumor cells/mL. Transduced T cells and non-transduced T cells wereadded to tumor cells at varying effector to target cell ratios in96-well tissue culture plates in a final volume of 200 uL, and incubatedfor 4 hours at 37° C. Thereafter, 40 uL of supernatant from each wellwas analyzed using Lumaplate-96 microplates (Packard Bioscience) by aTop Count NXT microplate scintillation counter (Packard Bioscience).Effector cell number in all CTL assays was calculated based on the totalnumber of T cells.

FIG. 10B shows the results for the Cr51 cytotoxic release assay in EL4tumor cells. Line 1 refers to transduced T cells applied to EL4 tumorcells expressing K9CD20; Line 2 refers to non-transduced T cells appliedto EL4 tumor cells expressing K9CD20; Line 3 refers to transduced Tcells applied to EL4 tumor cells that do not express K9CD20; and Line 4refers to non-transduced T cells applied to EL4 tumor cells that do notexpress K9CD20As shown in FIG. 10B, transduced T cells specificallykilled EL4 tumor cells expressing K9CD20 (see line number 1 in FIG. 10B)but not EL4 wild-type tumor cells (see Line 3 in FIG. 10B). In addition,non-transduced T cells did not kill EL4 tumor cells (see Lines 2 and 4in FIG. 10B).

FIG. 10C shows the results for the Cr51 cytotoxic release assay in NALM6tumor cells. Line 1 refers to transduced T cells applied to NALM6 tumorcells expressing K9CD20; Line 2 refers to non-transduced T cells appliedto NALM6 tumor cells expressing K9CD20; Line 3 refers to transduced Tcells applied to NALM6 tumor cells that do not express K9CD20; and Line4 refers to non-transduced T cells applied to NALM6 tumor cells that donot express K9CD20As shown in FIG. 10C, transduced T cells specificallykilled NALM6 tumor cells expressing K9CD20 (see line number 1 in FIG.10C) but not NALM6 tumor cells expressing Luciferin control (see Line 3in FIG. 10C). In addition, non-transduced T cells did not kill any tumorcells (see Lines 2 and 4 in FIG. 10C).

In Vivo NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wj1)/ScJ (NSG) mouse tumor models

6- to 8-week-old NSG mice were inoculated with 0.5×10⁶ NALM-6 tumorcells that expressed more than 95% luciferase-GFP and canine CD20-dsRedby tail vein injection each mouse (day 0). Mice were treated with 5×10⁶of CAR+ T cells (transduced group) and equal amount UT cells(untransduced group) by tail vein injection at day 4.

FIG. 11 shows that human T cells expressing K9CD20-targeted CAR arefunctional in vivo in a mouse model of lymphoma as the NALM-6 tumorcells expressing luciferase are eradicated over time in mice infusedwith K9CD20-CAR and expand in mice treated with untransduced T cells (asshown by luminescence upon infusion with luciferin). V: ventral view; D:Dorsal view.

Canine T-Cell Cultures, Activation and Retroviral Transduction

Blood samples were obtained from healthy canine donor. Peripheral bloodmononuclear cells (PBMC) were separated on Ficoll-Paque™ plus (GEhealthcare). Thawed or freshly isolated canine PBMCs were activated withPHA (Fisher Scientific) in RPMI (Corning) containing 15% FBS, 2 mmol/LL-glutamine (Life Technologies), 100 units/mL penicillin, 100 ug/mLstreptomycin (Life Technologies), and 50 to 200 units/ml of IL2 (R&DSystems) for 48 hours. Activated canine T cells were transduced inRetroNectin-coated 6-well plates with retroviral vector at the finaldensity of approximately 0.5×10⁶ cells/mL by spinoculation at 1200 rpmfor 1 hour. Transduction efficiency is evaluated 7 days posttransduction by qPCR analysis.

Cytotoxic Lymphocyte (CTL) Assay using Canine T Cells

Cytotoxic activity of transduced canine T cells was determined by 51 Crrelease assays as previously described (Yuan et al., J Immunol 176 (4),2006). Briefly, NALM-6 tumor cells were labeled with ⁵¹Cr for 1 hour at37° C., washed with RPMI medium supplemented with 10% FCS, andresuspended in the same medium at a concentration of 1×10⁵ tumorcells/mL. Transduced canine T cells or non-transduced canine T cellswere added to pre-labeled tumor cells at varying effector to target cellratios in 96-well tissue culture plates in a final volume of 200 uL andincubated for 4 hours at 37° C. 40 uL of supernatant from each well wassubsequently analyzed using Lumaplate-96 microplates (PackardBioscience) by a Top Count NXT microplate scintillation counter (PackardBioscience). CAR expression as well as CD4:CD8 ratio on the sameperforming CTL were assessed by specific antibodies using FACS analysis.Effector cell numbers in CTL assays were calculated based on the totalnumber of T cells.

FIG. 12 shows the results for the Cr51 cytotoxic release assay in NALM6tumor cells treated with canine T cells. Line 1 refers to non-transducedcanine T cells applied to NALM6 tumor cells expressing luciferin; Line 2refers to canine T cells transduced with the SFG-K27 CAR constructapplied to NALM6 tumor cells expressing luciferin; Line 3 refers tocanine T cells transduced with the SFG-K36 CAR construct applied toNALM6 tumor cells expressing luciferin; Line 4 refers to non-transducedcanine T cells applied to NALM6 tumor cells expressing K9CD20; Line 5refers to canine T cells transduced with the SFG-K27 CAR constructapplied to NALM6 tumor cells expressing K9CD20; and Line 6 refers tocanine T cells transduced with the SFG-K36 CAR construct applied toNALM6 tumor cells expressing K9CD20. As shown in FIG. 12 , transducedcanine T cells specifically killed NALM6 tumor cells expressing K9CD20antigen (NALM6-K9CD20) but not control NALM6 tumor cells expressingluciferin (NALM6-Luciferin). Canine T cells transduced with SFG-K27 CARexpress the K27 CAR comprising the anti-K9CD20scFv, K9-CD28 signalingdomain and K9-CD3z chain. Canine T cells transduced with SFG-K36 CARco-express the K27 CAR and 41BBL.

FIG. 13 shows the results for the Cr51 cytotoxic release assay in NALM6tumor cells treated with canine T cells. Line 1 refers to non-transducedcanine T cells applied to NALM6 tumor cells expressing luciferin; Line 2refers to canine T cells transduced with the SFG-K27 CAR constructapplied to NALM6 tumor cells expressing luciferin; Line 3 refers tocanine T cells transduced with the SFG-K36 CAR construct applied toNALM6 tumor cells expressing luciferin; Line 4 refers to canine T cellstransduced with the SFG-k9CD34t-K27 CAR construct applied to NALM6 tumorcells expressing luciferin; Line 5 refers to canine T cells transducedwith the SFG-k9CD34t-K36 CAR construct applied to NALM6 tumor cellsexpressing luciferin; Line 6 refers to non-transduced canine T cellsapplied to NALM6 tumor cells expressing K9CD20; Line 7 refers to canineT cells transduced with the SFG-K27 CAR construct applied to NALM6 tumorcells expressing K9CD20; Line 8 refers to canine T cells transduced withthe SFG-K36 CAR construct applied to NALM6 tumor cells expressingK9CD20; Line 9 refers to canine T cells transduced with theSFG-k9CD34t-K27 CAR construct applied to NALM6 tumor cells expressingK9CD20; and Line 10 refers to canine T cells transduced with theSFG-k9CD34t-K36 CAR construct applied to NALM6 tumor cells expressingK9CD20. As shown in FIG. 13 , transduced canine T cells specificallykilled NALM6 tumor cells expressing K9CD20 antigen (NALM6-K9CD20) butnot control NALM6 tumor cells expressing luciferin (NALM6-Luciferin).Canine T cells transduced with SFG-K27 CAR express the K27 CARcomprising the anti-K9CD20scFv, K9-CD28 signaling domain and K9-CD3zchain. Canine T cells transduced with SFG-K36 CAR co-express the K27 CARand 41BBL. Canine T cells transduced with SFG-K9CD34t-K27 express theK27CAR and the truncated K9CD34t. Canine T cells transduced withSFG-K9CD34t-K36 express the K36CAR and the truncated K9CD34t.

Example 5: Evaluation of Optimal Dose and Combination of CAR T CellTherapy

In this Example, the efficacy and safety profile of CAR T cell therapyin canines are evaluated. Dogs are treated with a CAR T cell therapycomprising K27CAR-4-1BBL T cells and/or K27CAR-PD1-DNR T cells. In a CART cell therapy, dogs are infused with CAR T cells. The efficacy of CAR Tcell therapy is measured by B cell lymphodepletion.

In addition, dogs may be treated with therapeutic regimens comprisingK27CAR-4-1BBL T cells and/or K27CAR-PD1-DNR T cells in combination withcyclophosphamide (Cy) and/or total body irradiation (TBI) (e.g., lowdose TBI, for example 0.01 Gy to 1.0 Gy). In the CAR T combinationtherapy, dogs may be treated with Cy at least 24 hours prior to thefirst T cell infusion. The CAR T combination therapy may be performed ina myeloablative setting. Alternatively, the CAR T combination therapy isperformed in a nonmyeloablative setting.

Methods for collecting T cells, transducing T cells, infusing CAR Tcells, and evaluating the efficacy and toxicity of CAR T cell therapyare described in this Example.

T Cell Collection

Prior to the T cell infusion, autologous T cells are collected from aleukapheresis product. Leukapheresis is performed for example on a COBESpectra machine. A single leukapheresis cell product should be enoughfor expansion and generation of autologous CAR T cells. Additionally,lymph node aspirates are collected to determine baseline disease.Additionally, lymph node aspirates are collected without anesthesiausing a 21- or 22-gauge needle.

T Cells Purification, Transduction and Ex Vivo Characterization

The collected T cells are separated on a Ficoll gradient, expanded, andactivated ex vivo. Briefly, canine PBMCs are stimulated with PHA andtransduced with K27/PD1-DNR or K27/41BBL CAR vectors pseudotyped withRD114 envelope using 50 to 200 U/ml of human IL-2 (hIL-2). Theleukapheresis product is characterized by flow cytometry analysis ofCD3+, CD4+, CD8+, CCR7+, CD62L+, CD28+, CD27+ cells to identify T cells.The expression of CD21 together with CD20 are evaluated to identify Bcells. CD20 expression is assessed with an 18F6 rat antibody from whichthe anti-K9CD20 ScFv was derived and which co-stains the majority of theCD21+ normal B cells in canine peripheral blood by FACS. The phenotypeof transduced CART cells and remaining B cells, if any, is subsequentlycharacterized by flow cytometry with the same antibodies in addition toCD34 (which is used as a surrogate for transduction efficiency).Functionality of CAR T cells is assessed in a 51 Cr release assay usingK9CD20-expressing target cells. Cytokine production in end-of-production(EOP) CAR T cells is evaluated upon restimulation with K9CD20 AAPCs(NIH3T3 fibroblasts expressing K9CD20) using the Luminex technology(CCYTOMAG-90K Milliplex EMD Millipore). CAR T cells can be infusedeither fresh or post-thaw.

CAR T Cell Infusion

Following ex vivo transduction, expansion and characterization, the CART cell product is infused intravenously in dogs. Depending on CAR T cellpersistence after the first infusion, each dog could receive a seconddose 2 to 4 weeks later as indicated in Table 7. For K27/PD1-DNR CAR Tcell therapy (also called 28z/PD1-DNR), the first infusion consists of10⁶ cells/kg and the second infusion consists of 3×10⁶ cells/kg. ForK27/41BBL CART cell therapy (also called 28z/41BBL), the first infusionconsists of 10⁵ cells/kg and the second infusion consists of 3×10⁵cells/kg. The same doses of both CAR-T cells are infused in the CAR Tcell combination therapy (also called Combo 28z/PD1-DNR 28z/41BBL)(Table 7 and FIG. 15 ).

TABLE 7 Dose escalation/de-escalation Dose T cell Dose T cell DoseNumber of Level 28z/PD1-DNR 28z/41BBL Combo Dogs −1 10⁶ CAR T 10⁵ CAR Tcells/kg 28z/PD1-DNR + 3 cells/kg 28z/41BBL 1 3 × 10⁶ CAR T 3 × 10⁵ CART 28z/PD1-DNR + 3 cells/kg cells/kg 28z/41BBL 2 10⁷ CAR T 10⁶ CAR Tcells/kg 28z/PD1-DNR + 3 cells/kg 28z/41BBL

Engraftment Monitoring

The engraftment of the infused CAR T cells is monitored in theperipheral blood and lymph nodes. Blood draws are performed on days 1,3, and 7 following T cell infusion, then once a week until CAR T cellsare not detected in two consecutive blood draws. Lymph node aspiratesare also collected 1, 2, and 4 weeks after the infusion to monitor CAR Tcell trafficking and persistence. The CAR T constructs are tagged withthe truncated canine CD34 (tCD34), allowing for CAR T cells tracking invivo. Flow cytometry analyses are performed on both peripheral blood andlymph nodes aspirates using canine CD45, CD3, CD4, CD8, CD34, CD62L,CD27, CCR7, and CD28 staining. Alternatively, if an insufficient numberof cells is obtained, especially in the lymph nodes, RNA is extracted toenable the detection of CAR T cells by quantitative PCR analysis(utilizing PCR primers specific for the junction with 41BBL or PD1-DNR).If T cell numbers allow, retrieved CAR T cells are analyzed by RNA Seq(see Example 7). A leukapheresis is performed at day 28 to provideadditional material for complete functional (cytotoxicity, cytokinesecretion), flow cytometry (differentiation and exhaustion phenotype),and genomic (vector copy number, RNAseq) studies (see Example 7).

Determination of Maximum Tolerated Doses and Toxicities

Toxicities are closely monitored. To this end, lymphodepletion and, morespecifically, the kinetics of B cell aplasia as well as recovery areevaluated by flow cytometry detection of CD21+ cells at the same timepoints as indicated (FIG. 15 ) in both peripheral blood and popliteallymph nodes. B cell aplasia may be indirectly monitored by serumimmunoglobulin quantified by serum protein electrophoresis.Additionally, severe CRS has been characterized in humans by the releaseof a variety of cytokines such as interleukin-6 (IL-6), interferon-y,and IL-10 in the serum of patients experiencing fever, tachycardia, andhypotension symptoms. Such increase in cytokines release has also beenassociated with B cell lymphoma in dogs and is quantified in the serumat each blood draw using Luminex technology (EMD Millipore Milliplex)with a special emphasis on IL-6, interferon-y and TNFα and correlatedwith toxicities and adverse events. To resolve AEs, steroids such asdexamethasone may be injected to curtail the expansion of CAR T cells.In the event of neurotoxicity following T cell infusion, CSF iscollected and analyzed for presence of CART cells and cytokines. CRPlevels are measured daily until the CAR T cell expansion riches aplateau using the LifeAssays® Canine CRP Test to determine if it mayserve as a predictor of severe cytokine release syndrome (sCRS). Serumimmunoglobulin and serum protein electrophoresis.

Statistical Considerations

Two CAR T cell constructs (K27/41BBL and K27/PD1-DNR) may be tested,each individually and upon co-infusion of both subsets of CART cells.The primary objective for each group is to identify a dose that is safeand efficacious. Efficacy is assessed by the appearance of B-cellaplasia and safety is defined as lack of sCRS. Any occurrence of sCRSresults in de-escalation of the dose for one or the combined two CAR Tcell constructs. If the occurrence of B-cell aplasia is not frequentenough, then the dose of CAR T cells is escalated to the next higherdose level. Only two dose levels are considered unless sCRS occurs atthe starting dose level, in which case the dose is de-escalated.

Conditions to be considered for identifying whether a dose hassufficient efficacy and an acceptable toxicity profile include thefrequency of occurrence of sCRS and B cell aplasia.

Example 6: Determining the Efficacy of Lymphodepletion and CAR Design inCompanion Dogs with CD20+B Cell Lymphoma

In this Example, the functional persistence, safety, and efficacy ofautologous CAR T cells expressing either K27/PD1-DNR or K27/41BBL and/orboth subsets in canine companion patients with B cell lymphoma (n=9 to15) using the optimal dosing identified in Example 5 are evaluated.Correlative studies focus on B cell aplasia and recovery, tumoreradication, CAR T cell persistence, functionality, and exhaustion, andthe impact of CAR T cells on endogenous lymphoid and myeloid cells (seeExample 7).

Dog Studies and Chemotherapy

Similar to the experimental animals in Example 5, companion dogs aretreated with Cy prior to CAR T cell infusion. As possible, blood drawsand lymph nodes aspirates are performed on the same schedule asdescribed in Example 5 and FIG. 15 , in order to monitor CAR T cellpersistence.

Enrollment criteria is similar to human patients treated with CD19targeted CAR T cells: Patient dogs must have B cell lymphoma that hasrelapsed after a response to at least one prior therapy regimen or isrefractory to prior therapy. Patient dogs come to the laboratory forapheresis and then return to their owner or referring veterinarianphysicians. After CAR T cells have been generated, dogs return to thelaboratory and receive Cy as conditioning as discussed in Example 5. Atleast 24 hours after Cy, the patient dogs receive the CAR T cells withappropriate premedication. Animals return to the veterinary clinic forclose observation and monitoring for cytokine release syndrome (CRS).Disease is assessed by analysis of CD79a, IgM, and/or CD21 expression onlymph node aspirates when possible. The expression of CD20 is alsoevaluated using the antibody 18F6, from which the anti-K9CD20 ScFv wasderived and which co-stains most of the CD21+normal B cells in canineperipheral blood.

FIG. 24 shows flow cytometry data demonstrating detection canine CD20with antibody 18F6. The figure shows Canine PBMCs cells stained withCD21 (PE/FL2-H) alone or in combination with antibody 18F6.

CAR T Cells Generation and Infusion

Autologous T lymphocytes are obtained through Ficoll density gradient,activated, and expanded ex vivo as described in Example 4. Following thesame experimental procedure as described in Examples 4 and 5, the cellsare transduced with CAR construct(s) described in Example 5 with themost efficient and least toxic dose identified in Example 5. Theleukapheresis and generated CAR T cells are characterized by flowcytometry analysis with the same antibody panels as described in Example5. A ⁵¹chromium release assay and cytokine quantification may beperformed as described in Example 5.

Engraftment Monitoring

CAR T cell persistence is analyzed on peripheral blood and lymph nodesamples collected as described in Example 5. T cell infusion engraftmentis monitored at each blood draw at days 1, 3, and 7 following the T cellinfusion, then once a week until CAR T cells are not detected in twoconsecutive blood draws. Additionally, CAR T cells trafficking to andpersistence in the lymph nodes are evaluated at 1, 2, and 4 weekspost-infusion. Infused CAR T cells can be tracked in vivo by staining ofthe truncated canine CD34 (and corroborated by qPCR for vectorsequences). The phenotype of T cells is monitored using CD3, CD4, CD8,CD34, CCR7, CD62L, CD28, CD27 staining. If T cell numbers allow,retrieved CAR T cells are analyzed by RNA Seq. A leukapheresis isperformed at day 28 to provide additional material for completefunctional (cytotoxicity, cytokine secretion), flow cytometric(differentiation and exhaustion phenotype), and genomic (vector copynumber, RNAseq) studies.

Toxicities

The kinetics of B cell aplasia versus recovery is monitored at the timepoints indicated in FIG. 15 by flow cytometry analysis of CD20 (with18F6 antibody), CD21+ cells in the peripheral blood, plus lymph nodesaspirates at weeks 1, 2, and 4 post-infusion. Serum immunoglobulin,serum protein electrophoresis, cytokines in the serum for each blooddraw of each patient, CRP levels are measured as described in Example 5at the same time points. To resolve AEs, steroids such as dexamethasonemay be injected to curtail the expansion of CAR T cells. In the event ofneurotoxicity, CSF is analyzed for presence of CAR T cells andcytokines.

Tumor Eradication

Tumor eradication is evaluated based on the disappearance of clinical(physical evaluation) and cellular evidence of leukemic cells (flowcytometry with CD79a, IgM CD21, and/or 18F6 antibodies), restoration ofnormal hematopoiesis, as well as serum concentrations of cytokines.

Example 7: Comprehensive Correlative Studies to Evaluate Safety andEfficacy of CD20 CAR T Cell Therapy in Companion Dogs

The main focus of this Example is to determine the action of either28z/PD-1 DNR or 28z/4-1BBL CAR T cells alone or in combination onendogenous T cells and on the tumor microenvironment. Specifically, thisExample focuses on a) the functional persistence of infused CAR T cells,b) cytokine responses in vitro in EOP CAR T cells, in vivo in peripheralblood and in CRS, and on c) the trans-costimulatory effect ofconstitutive 4-1BBL expression in CAR T cells and CAR T cell cytokinesecretion is assessed in tumor specimens (bone marrow or lymph node). Inaddition to enumerating tumor cells, T cells (CAR T cells, non-CAR Tcells, and Tregs) and myeloid cells, the level of expression of forexample PD-L1 and HLA class I on tumor cells and surrounding cellsincluding Tregs and myeloid cells are examined. Peripheral blood andserum are collected at 1, 3, 7 days and weekly thereafter for analysis.Lymph node aspirates are collected pretreatment and at 1, 2 and 4 weekspost infusion as possible. Apheresis is collected 28 days after CAR Tcell infusion.

Flow Cytometric Studies and qPCR Assay

CAR, B, T and myeloid lineage cells' phenotypes are examined asdescribed in Examples 5 and 6. T cell peak expansion (PK) andpersistence are measured by FACS analysis and corroborated by qPCRmeasuring the average vector copy number (VCN) in tissues samples. TheqPCR assay is designed to distinguish K27/PD1-DNR and K27/41BBL and isperformed on cells from CSF. If T cell numbers allow, RNA is performedat these time points and at day 28 (apheresis).

Cytokine Studies and CSF Analyses

Multiple cytokines and chemokines are monitored using (CCYTOMAG-90KMilliplex EMD Millipore) and read on Luminex. Cytokines are monitored incanine serum, plasma pre- and post-infusion, in CSF and in EOP CAR Tcells prior to infusion and upon restimulation in vitro. If possible(especially in day 28 apheresis), CART cells are selected based on theexpression of truncated CD34 molecule to conduct these assays. Post CART cell infusion, pro-inflammatory cytokines, such as IL-6, aremonitored. The cytokine profiles between K27CAR/PD1-DNR andK27CAR/41BBL-treated dogs are compared.

CRP Monitoring

CRP levels are measured daily until the CAR T cell expansion reaches aplateau using the LifeAssays® Canine CRP Test to determine if it mayserve as a predictor of severe CRS.

RNAseq/Gene Expression Profiling

RNA seq technology is used to characterize the tumor, the tumorenvironment and the host immunologic responses to CAR T cells. RNAseq isperformed. Lymph nodes aspirates are collected before treatment and 1, 2and 4 weeks post infusion, 4 weeks being the time point by which mostresponses are observed in clinical trials using human 1928z CAR T cells.RNA extracted from isolated cells are submitted to whole genomesequencing. Gene expression profile is analyzed using the CanFam3.1Assembly of the dog genome that encompasses −15,000 annotated genetranscripts is regularly updated. The RNAseq assay is used to inform onthe detection of CAR T cells in the tumor. The analysis focuses onidentifying genes in which transcription is altered after infusion ofCAR T cells. Gene expression profile results are compared betweenresponders and non-responders and used to identify factors in the tumor,the tumor environment, or infiltrating immune cells that impact clinicaloutcome. A similar analysis of peripheral blood leucocytes is undertakenat the same time points to study immune responses more broadly.

TCRseq

To elucidate the role of the native T cell response in establishing andmaintaining clinically sustainable anti-tumor immunity under CARtherapy, high-throughput T cell receptor sequencing (TCRseq) is used tostudy the canine TCR repertoire throughout treatment with K27CAR/PD1-DNRand K27CAR/41BBL CAR T cells. TCRseq allows bulk profiling of thecomplementarity-determining region 3 (CDR3) sequences that are generateduniquely in each T cell and clonally expanded in the population uponantigen stimulation. The C-domains of both the T cell receptor alpha(TRA) and T cell receptor beta (TRB) loci are well mapped, making bothchains accessible to TCRseq library generation using universalamplification methods. Reagents are used to amplify and sequence thecanine TRA/TRB (and eventually TRG/TRD) loci.

TCRseq is used to determine the extent of epitope spreading duringresponse to CAR T cell therapy, and whether the additionaltrans-stimulatory effects of PD1-DNR and 4-1BBL facilitate epitopespreading to provide a therapeutic benefit. TCRseq of the caninerepertoire, combined with the computational and analytical resources, isused to determine the timing, strength, and breadth of the induction ofantigen-driven clonal/oligoclonal expansion of native canine T cells inthe presence of CAR-T activity, and evaluate the impact of 4-1BBLstimulation on this process.

What is claimed is:
 1. A method of treatment comprising a. isolatingT-cells from a subject, b. transfecting the T-cells with a vectorcomprising a nucleic acid encoding an isolated antibody or an antigenbinding portion thereof that specifically binds to a canine CD20 cyclicpeptide having the sequence of SEQ ID NO: 20, wherein the cysteine atposition 7 of SEQ ID NO: 20 forms a disulfide bond with the cysteine atposition 23 of SEQ ID NO: 20, and c. administering the transfectedT-cells to the subject.
 2. The method of claim 1, wherein the subject isa canine subject.
 3. The method of claim 1, wherein the antibody orantigen binding portion thereof binds to the canine CD20 cyclic peptideat a higher affinity than a linear peptide having the sequence of SEQ IDNO:
 21. 4. The method of claim 1, wherein the antibody or antigenbinding portion thereof comprises a heavy chain variable domain (VH)comprising SEQ ID NO: 4, or is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO:
 4. 5. The method of claim 1, whereinthe antibody or antigen binding portion thereof comprises a VHcomplementarity determining region (CDR)1 of SEQ ID NO: 40, or is atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NO:
 40. 6. The method of claim 1, wherein the antibody orantigen binding portion thereof comprises a VH CDR2 of SEQ ID NO: 42, oris at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO:
 42. 7. The method of claim 1, wherein theantibody or antigen binding portion thereof comprises a VH CDR3consisting of a threonine (T) residue.
 8. The method of claim 1, whereinthe antibody or antigen binding portion thereof comprises a light chainvariable domain (VL) comprising SEQ ID NO: 6, or is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
 6. 9. The methodof claim 1, wherein the antibody or antigen binding portion thereofcomprises a VL CDR1 of SEQ ID NO: 46, or is at least 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
 46. 10. Themethod of claim 1, wherein the antibody or antigen binding portionthereof comprises a VL CDR2 of SEQ ID NO: 48 or a VL CDR2 having atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with SEQ ID NO:
 48. 11. The method of claim 1, whereinthe antibody or antigen binding portion thereof comprises a VL CDR3 ofSEQ ID NO: 50 or a VL CDR3 having at least 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 50.12. The method of claim 1, wherein the antibody or antigen bindingportion thereof is a full length antibody, a Fab fragment, a F(ab′)2fragment, or a single chain variable fragment (scFV).
 13. The method ofclaim 1, wherein the antibody is a scFv.
 14. The method of claim 1,wherein the antibody or antigen binding portion thereof comprises SEQ IDNO: 8, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO:
 8. 15. The method of claim 1, wherein the antibody or antigenbinding portion thereof is a chimeric antigen receptor (CAR).